Anti-connexin compounds targeted to connexins and methods of use thereof

ABSTRACT

Methods and compositions for modulating the activities of connexins are provided, including, for example, for use in post-surgical, trauma, or tissue engineering applications. These compounds and methods can be used therapeutically, for example, to reduce the severity of adverse effects associated diseases and disorders where localized disruption in direct cell-cell communication is desirable.

RELATED PATENT APPLICATIONS

This application is a divisional application of and claims benefit ofpriority under 35 U.S.C. §119(e) to U.S. Utility application Ser. No.13/230,744, entitled “ANTISENSE COMPOUNDS TARGETED TO CONNEXINS ANDMETHODS OF USE THEREOF,” filed on Sep. 12, 2011, which is a divisionalof U.S. Utility application Ser. No. 10/581,813, entitled “ANTISENSECOMPOUNDS TARGETED TO CONNEXINS AND METHODS OF USE THEREOF,” filed onOct. 11, 2011, which claims benefit of National Stage Application under35 U.S.C. §371 of International Application No. PCT/IB2004/004431,entitled “ANTISENSE COMPOUNDS TARGETED TO CONNEXINS AND METHODS OF USETHEREOF,” filed on Dec. 3, 2004, which claims the benefit of New ZealandApplication No. 529936, entitled “TISSUE ENGINEERING OR REMODELING,”filed on Dec. 3, 2003, each of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present disclosure relates to and describes agents, compositions andmethods of using compounds for modulation of gap-junction-associatedprotein expression. These agents, compositions, and methods are useful,for example, for tissue engineering in vivo and in vitro, including forexample in the skin, in corneal tissue, and conjunction with surgicalprocedures of the eye.

BACKGROUND

Tissue or organ failure due to illness or injury is a major healthproblem worldwide with little option for full recovery other than organor tissue transplantation. However, problems finding a suitable donormean that this option is not available to the majority of patients.tissue engineering or remodeling whereby synthetic or semi synthetictissue or organ mimics that are either fully functional or which aregrown in a desired functionality is currently being investigated asreplacements.

One area in particular that this technology is becoming increasinglyimportant is in the cornea of the eye. Corneal transplantation is themost common form of solid organ transplant performed worldwide. Eachyear around 80,000 are performed in the USA and the UK alone. Theprevalence of refractive surgery for correction of myopia such asphotorefractive keratectomy (PRK) and laser in situ keratomileusis(LASIK) has led to shortage of suitable cornea for transplant for tissuereconstruction after surgery or disease processes and for tissuemanipulation in vivo to engineer changes. In addition, approximately 5%of patients undergoing laser surgery experience unexpected outcomes.

The cornea is a transparent tissue that comprises the central one sixthof the outer tunic of the eye. Its unified structure and functionprovide the eye with a clear refractive interface, tensile strength, andprotection from external factors. The cornea is built from threedifferent main layers of cells: the epithelium, the stroma, and theendothelium (Pepose, J. S. et al., “The cornea; Adler's Physiology ofthe eye: Clinical application”, 9^(th) ed. St. Louis: Mosby Year Book,1992, 29-47; Spencer, W. H., “The cornea; Ophthalmic Pathology: an atlasand textbook”, 4^(th) ed., Philadelphia: W. B. Saunders Co., 1996,157-65). In addition, the Descemet's membrane, the Bowman's layer, andthe basement membrane are structures that are derived in some ways fromone of these main cellular layers.

The corneal epithelium is the layer in direct contact with the externalenvironment. It is a stratified squamous, non-keratinized structure witha thickness ranging from 40 to 100 μm, in rats and in humans,respectively. It is comprised of a superficial zone, usually formed bytwo to three layers of flat squamous cells; a middle zone, formed by twoor three layers of polyhedral wing cells; and a basal zone consisting ofa single row of columnar cells. The stratified corneal epithelium ischaracterized as a “tight” ion transporting functional syncitium whichserves both as a protective barrier to the ocular surface, as well as anadjunct fluid secreting layer assisting the corneal endothelium in theregulation of stromal hydration, and thereby contributing to themaintenance of corneal transparency. The unique and specializedqualities offered by the corneal epithelium have been proven to beessential for the operation of the cornea as the principal refractiveelement of the eye. It is therefore important that its stratifiedstructure be maintained irrespective of any environmental stresses.

Trauma to the surface of the cornea is highly prevalent; for example,minor scrapes, eye infections and diseases, chemical or mechanicalaccidents and surgical practice can all damage the cornea. One majorcomplication in post corneal-trauma wound healing is the loss of visualacuity due to tissue reorganization. Patients at risk for ophthalmichealing problems include those who have undergone surgery. Examples ofsuch surgery include, but are not limited to, cataract extraction, withor without lens replacement; corneal transplant or other penetratingprocedures, such as penetrating keratoplasty (PKP); excimer laserphotorefractive keratectomy; glaucoma filtration surgery; radialkeratotomy; and other types of surgery to correct refraction or replacea lens.

The cornea provides the external optically smooth surface to transmitlight into the eye. Surgery disrupts the forces which anchor the corneain its normal configuration. In cataract patients, a full-thicknesssurgical incision is made in the region of the limbus. The corneacontracts when it heals, causing a local distortion of the tissue and aconcomitant distortion in the visual field in the affected region(astigmatism).

Other surgical wounds in the cornea can initiate a wound healing processthat causes a predetermined local shift in the curvature of the cornea.The most widely known of these techniques is radial keratotomy (RK), inwhich several partial-thickness incisions are produced to cause centralcorneal flattening. This technique, however, is limited due to a lack ofpredictable results and significant fluctuations in vision, both ofwhich are related to the nature and extent of wound healing (Jester etal., Cornea (1992) 11: 191). For example, a reduction in peripheralbulging of the corneal tissue with an associated regression in theinitial visual improvement has been observed in most RK patients(McDonnell and Schanzlin, Arch. Ophthalmol. (1988), 106: 212).

Wounds in the cornea also heal slowly, and incomplete healing tends tobe associated with instability of visual acuity (with fluctuations invision from morning to evening, as well as drifting visual acuityoccurring over a period of weeks to months). This may be the cause of34% or more of patients who have had radial keratotomy complaining offluctuating vision one year after surgery (Waring et al., Amer. J.Ophthalmol. (1991) 111: 133). Also, if a corneal wound fails to healcompletely, a wound “gape” can occur leading to a progressive hyperopiceffect. Up to 30% of patients having the RK procedure are afflicted withhyperopic shifts associated with wound gape (Dietz et al., Ophthalmology(1986) 93: 1284).

Corneal regeneration after trauma is complex and not well understood. Itinvolves the regeneration of three tissues: the epithelium, the stromaand the endothelium. Three main intercellular signaling pathways arethought to coordinate tissue regeneration: one mediated by growthfactors (Baldwin, H. C. and Marshall, J., Acta Ophthalmol. Scand.,(2002) 80: 238-47), cytokines (Ahmadi, A. J. and Jakobiec, F. A., Int.Ophthalmol. Clinics, (2002) 42(3): 13-22) and chemokines(Kurpakus-Wheater, M, et al., Biotech. Histochem, (1999) 74: 146-59);another mediated by cell-matrix interactions (Tanaka, T., et al., Jpn.J. Ophthalmol., (1999) 43: 348-54); and another mediated by the gapjunctions and the connexin family of channel forming proteins.

Gap junctions are cell membrane structures, which facilitate directcell-cell communication. A gap junction channel is formed of twoconnexins, each composed of six connexin subunits. Each hexamericconnexin docks with a connexin in the opposing membrane to form a singlegap junction. Gap junction channels can be found throughout the body. Atissue such as the corneal epithelium, for example, has six to eightcell layers, yet expresses different gap junction channels in differentlayers with connexin-43 in the basal layer and connexin-26 from thebasal to middle wing cell layers. In general, connexins are a family ofproteins, commonly named according to their molecular weight orclassified on a phylogenetic basis into alpha, beta, and gammasubclasses. To date, 20 human and 19 murine isoforms have beenidentified (Willecke, K. et al., Biol. Chem., (2002) 383, 725-37)perhaps indicating that each different connexin protein may befunctionally specialized. Different tissues and cell types havecharacteristic patterns of connexin protein expression and tissues suchas cornea have been shown to alter connexin protein expression patternfollowing injury or transplantation (Qui, C. et al., (2003) CurrentBiology, 13: 1967-1703; Brander et al., (2004), J Invest Dermatol.122(5): 1310-20).

The corneal regeneration process post-trauma can result in the loss ofcorneal clarity and therefore influence the outcome of refractivesurgery. Present treatments for damaged cornea generally include cornealtransplant or attempts to use corneal cells/tissue for reconstruction.However, post-operative trauma to the corneal and the surrounding softtissue following surgical procedures such as, for example, excimer laserphotorefractive keratectomy, often results in scarring due tohypercellularity associated with modification of the extracellularmatrix; including changes in epithelial cell patterning, myofibroblastdifferentiation, stromal remodeling, and epithelial hyperplasia at thesite of a laser induced lesion.

In severe spinal cord injuries, the pathological changes that occur,whether by transection, contusion or compression, share somesimilarities with post-operative scar formation and tissue remodeling.Within 24-48 hours after injury, the damage spreads and significantlyincreases the size of the affected area. A gap junction-mediatedbystander effect (Lin, J. H. et al., 1998, Nature Neurosci. 1: 431-432),by which gap junction channels spread neurotoxins and calcium waves fromthe damage site to otherwise healthy tissue may be involved. This isaccompanied by the characteristic inflammatory swelling. The region ofdamage in the spinal cord is replaced by a cavity or connective tissuescar, both of which impede axonal regeneration (McDonald, J. W. et al,(September 1999) Scientific American. 55-63; Ramer, M. S. et al., SpinalCord. (2000) 38: 449-472; Schmidt, C. E. and Baier Leach, J.; (2003)Ann. Rev. Biomed. Eng. 5: 293-347). Although progress has been made withsome current therapeutic modalities, major constraints to spinal cordrepair still remains, including the invasive intervention itself canfurther lesion spread and glial scar formation, impeding the repairprocess and risk further loss of neural function (Raisman, G. J. RoyalSoc. Med. 96: 259-261).

Antisense technology has been used for the modulation of the expressionfor genes implicated in viral, fungal and metabolic diseases. U.S. Pat.No. 5,166,195, proposes oligonucleotide inhibitors of HIV. U.S. Pat. No.5,004,810 proposes oligomers for hybridizing to herpes simplex virusVmw65 mRNA and inhibiting replication. See also WO00/44409 to Becker etal., filed Jan. 27, 2000, and entitled “Formulations ComprisingAntisense Nucleotides to Connexins”, the contents of which are herebyincorporated by reference in their entirety, describes the use ofantisense (AS) oligodeoxynucleotides to downregulate connexin expressionto treat local neuronal damage in the brain, spinal cord or optic nerve,in the promotion of wound healing and reducing scar formation of skintissue following surgery or burns. However, many difficulties remainthat need to be overcome. It is often the case, for example, that thedown regulation of a particular gene product in a non-target cell typecan be deleterious. Additional problems that need to be overcome includethe short half life of such ODN's (unmodified phosphodiester oligomers)typically have an intracellular half life of only 20 minutes owing tointracellular nuclease degradation (Wagner 1994, supra) and theirdelivery consistently and reliably to target tissues.

Therefore, there is a need and there are enormous potential advantagesfor the development of compounds for the problems described above. Suchcompounds, related compositions, and methods for their use are describedand claimed herein.

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Summary. The inventions described and claimedherein are not limited to or by the features or embodiments identifiedin this Summary, which is included for purposes of illustration only andnot restriction.

Provided herein are compounds useful for tissue engineering, includingantisense compounds. Also provided are antisense compounds and methodsfor reducing tissue damage associated with ophthalmic procedures. Themethods comprise, for example, administering an antisense compound tothe eye of a subject in an amount sufficient to inhibit the expressionof a connexin protein in the eye or in cells associated with the eye ofthe subject. While it is preferred that the expression of connexinprotein is inhibited, it is envisioned that other proteins may betargets for modulation by the compounds, including the antisensecompounds, either alone of in combination with antisense or othercompounds that inhibit the expression of human connexins.

In certain embodiments, the ophthalmic procedure is an ophthalmicsurgery, including but not limited to an excimer laser photorefractivekeratectomy, a cataract extraction, corneal transplant, a surgery tocorrect refraction, a radial keratotomy, a glaucoma filtration surgery,a keratoplasty, an excimer laser photorefractive keratectomy, a cornealtransplant, a surgery to correct refraction, a ocular surface neoplasmexcision, a conjunctival or amniotic membrane graft, a pterygium andpingeculae excision, a ocular plastic surgery, a lid tumour excision, areconstructive lid procedures for congentital abnormalities, anectropian and entropian eyelid repair, a strabismus surgery (occularmuscle), or any penetrating eye trauma.

Generally, at least a portion of the nucleotide sequence is known forconnexins in which the inhibition of expression is desired. Preferably,an antisense compound is targeted to one or more specific connexinisotypes. Specific isotypes of connexins that may be targeted by theantisense compounds include, without limitation, 43, 37, 31.1, and 26.It is preferred, but not required, that the targeted connexins arehuman. A connexin (e.g., human) may, for example, have a nucleobasesequence selected from SEQ ID NO: 12-31.

In certain embodiments, antisense compounds are targeted to at leastabout 8 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO: 12-31.

In certain other embodiments, a second antisense compound isadministered to the subject (e.g. the eye), wherein one or more otherantisense compounds are targeted to at least about 8 nucleobases of anucleic acid molecule encoding a connexin (e.g., human) having anucleobase sequence selected from SEQ ID NO: 12-31. At least a secondantisense compound may, for example, be targeted to a different connexinthan a first antisense compound.

Examples of types of antisense compounds that may be used in variousaspects of the invention include antisense oligonucleotides, antisensepolynucleotides, deoxyribozymes, morpholino oligonucleotides, dsRNA,RNAi molecules, siRNA molecules, PNA molecules, DNAzymes, and5′-end-mutated U1 small nuclear RNAs, analogs of the preceding; as wellother compounds provided herein or known in the art; including but notlimited to, for example, non-specific uncouplers such as octanol,glycerhetinic acids, and heptanol.

In certain embodiments, for example, the antisense compounds areantisense oligonucleotides that comprise naturally occurring nucleobasesand an unmodified internucleoside linkage. In other embodiments, forexample, the antisense compounds are antisense oligonucleotidescomprising at least one modified internucleoside linkage, includingthose with a phosphorothioate linkage. Suitable antisense compounds alsoinclude, for example, oligonucleotides comprising at least one modifiedsugar moiety. Suitable antisense compounds also include, by way ofexample, oligonucleotides comprising at least one modified nucleobase.

In certain embodiments, antisense compounds provided herein areadministered in combination with another compound, for example acompound useful for reducing tissue damage, reducing inflammation,promoting healing, or some other desired activity.

In another aspect, the invention includes methods of treating a subject(e.g., a patient) by administering antisense compounds to the subject.

In certain embodiments, antisense compounds provided herein areadministered by local or topical administration. Antisense compoundsprovided herein can also be administered, for example, systemically orby intraocular injection.

Antisense compounds provided herein can be administered to a subject ata predetermined time, for example, relative to the formation of a wound,such as that occurs in an ophthalmic procedure (e.g., surgical). Forexample, antisense compounds can be administered before an ophthalmicprocedure is performed, during an ophthalmic procedure, or after anophthalmic procedure. Antisense compounds, for example, may beadministered to a subject within minutes or hours before or after anophthalmic procedure is performed. In certain embodiments, an antisensecompound is administered after an ophthalmic procedure is performed, andfor example the antisense compound is administered within about 4 hoursof the procedure, within about 3 hours of the procedure, and moretypically within about 2 hours of the ophthalmic procedure, or withinabout 1 hour of an ophthalmic procedure.

In another aspect, antisense compounds provided herein may beadministered in a methods to effect tissue engineering. For example,antisense compounds provided herein may be administered in conjunctionwith a method that increases the thickness of cornea tissue in asubject. Such method may, or may not, be associated with an ophthalmicprocedure (e.g., surgery). As an example, antisense compounds providedherein may be administered in conjunction with a method that promoteshealing or prevents tissue damage in cells associated with the cornea ofthe subject (e.g., corneal cells).

In certain embodiments, for example, the antisense compound decreasesscar formation. In certain embodiments, for example, the antisensecompound reduces inflammation. In certain embodiments, for example, theantisense compound promotes wound healing.

In certain preferred embodiments, for example, the antisense compound isused in association with a surgical implantation procedure.

In certain embodiments, for example, the antisense compound is directedto connexin 43 and is administered to regulate epithelial basal celldivision and growth.

In certain embodiments, for example, the antisense compound is directedto connexin 31.1 and is administered to regulate outer layerkeratinisation.

According to certain embodiments, for example, the ophthalmic procedureis cataract extraction. In other embodiments, for example, theophthalmic procedure is a corneal transplant. In other embodiments, forexample, the ophthalmic surgical procedure is surgery to correctrefraction. In another embodiments, for example, the ophthalmicprocedure is radial keratotomy. In another embodiments, for example, theophthalmic procedure is glaucoma filtration surgery. In still otherembodiments, for example, the ophthalmic procedure is keratoplasty. Inother embodiments, for example, the ophthalmic procedure is an ocularsurface neoplasm excision. In other embodiments, for example, theophthalmic procedure is a conjunctival or amniotic membrane graft. Inother embodiments, for example, the ophthalmic procedure is a pterygiumand pingeculae excision. In other embodiments, for example, theophthalmic procedure is an ocular plastic surgery. In other embodiments,for example, the ophthalmic procedure is a lid tumour excision. In otherembodiments, for example, the ophthalmic procedure is a reconstructivelid procedure for congentital abnormalities. In other embodiments, forexample, the ophthalmic procedure is an ectropian and entropian eyelidrepair. In other embodiments, for example, the ophthalmic procedure is astrabismus surgery (occular muscle). In other embodiments, for example,the ophthalmic procedure is a penetrating eye trauma.

In certain further embodiments, for example, compounds and compositionsare used to promote healing or to prevent tissue damage in cellsassociated with cornea, where the cells associated with the cornea maybe any cell in the eye, including but not limited to corneal cells.

The agents provided herein, including antisense compounds, may increasethe thickness of cornea tissue in a subject. In certain embodiments, forexample, the antisense compound is used in combination with anothercompound useful for reducing tissue damage or promoting healing. Forexample, the antisense compounds may be coadministered with a growthfactor, cytokine, or the like.

In another aspect, for example, a pharmaceutical composition forreducing tissue damage associated with ophthalmic surgery is provided.The pharmaceutical composition is suitably formulated, for example, fortopical or local administration to the eye of a subject comprising anantisense compound present in an amount sufficient to inhibit theexpression of a human connexin protein in cells associated with the eyeof the subject. The antisense compound, for example, is preferablytargeted to at least about 8 nucleobases of a nucleic acid moleculeencoding a connexin (e.g., human) having a nucleobase sequence selectedfrom SEQ ID NO:12-31.

In certain embodiments, for example, the antisense compounds are in theform of a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or vehicle and the agent or antisense compound ispresent in an amount effective to promote wound healing in a subject. Incertain embodiments, the pharmaceutical compositions may be, forexample, in a form suitable for topical administration, including in aform suitable for topical or local administration to the eye of asubject. In certain further embodiments, for example, the compositionsand formulations may be in the form of a gel, a cream, or any of theforms described herein or known in the art, whether currently or in thefuture.

In another aspect, the invention includes pharmaceutical compositionscomprising antisense compounds. In one embodiment, for example, apharmaceutical composition is provided for reducing tissue damageassociated with an ophthalmic procedure (e.g., surgery), such that thepharmaceutical composition is formulated for topical or localadministration to the eye of a subject and it comprises an antisensecompound present in an amount sufficient to inhibit the expression of ahuman connexin protein in cells associated with the eye of the subject.In certain embodiments, for example, the antisense compound is targetedto at least about 8 nucleobases of a nucleic acid molecule encoding aconnexin (e.g., human) having a nucleobase sequence selected from SEQ IDNO: 12-31.

In certain embodiments, for example, the pharmaceutical compositionincludes a pharmaceutically acceptable carrier comprising a bufferedpluronic acid or gel. This includes in one embodiment, for example, upto about 30% pluronic acid in phosphate buffered saline.

In another aspect, methods of designing antisense oligonucleotides thatare targeted to one or more connexin are provided. The method mayinclude the optimization of selected parameters, such as the thermostability, affinity, and specificity of a particular oligonucleotidewith a selected target. This method may be used to selected and developantisense oligonucleotides comprising one or more particular desiredpolynucleotide sequence. Testing of the antisense oligonucleotides maybe performed in conjunction with the method, for example, for theirability to cleave mRNA or block the translation of a connexin protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in vivo confocal microscopic images of corneas 12 hourspost photorefractive keratectomy in control and antisenseoligonucleotides treated eyes.

FIG. 2 shows histological examination of corneal remodeling in control(2A, 2B, 2C) and antisense oligodeoxynucleotide treated rat corneas (2D,2E) 24 hours after excimer laser photoablation.

FIG. 3 provides micrograph images showing expression of connexin 43protein in control (3A, 3B, 3C) and antisense oligodeoxynucleotidetreated corneas (3D, 3E, 3F) at 24 hours after excimer laser ablation.The results demonstrate that connexin43 protein levels are reducedfollowing treatment with anti-connexin43 ODNs and results in a smallerdegree of cell recruitment in the stroma.

FIG. 4 shows myofibroblast labeling 1 week after surgery using alphasmooth muscle actin antibodies. FIGS. 4A, 4B, and 4C are controls; and4D, 4E, and 4F are antisense treated corneas.

FIG. 5 shows laminin-1 labeling of control (5A, 5B) and connexin 43antisense oligodeoxynucleotide treated corneas 24 hours (5A-5D) and 48hours (5E-5H) after photorefractive keratectomy. At 24 hours controlshad little and/or uneven laminin deposition at the edge of the ablatedarea (5A) and more centrally (5B) whereas antisense treated corneasshowed a more regular deposition of laminin at both of these regions(5C, 5D). At 48 hours controls still do not have a continuous laminindeposition (5E—edge of the ablated area; FIG. 5F—central) and it wasvery uneven (5E). In contrast antisense ODN treated corneas had acontinuous and relatively even basal lamina at the wound edge (5G) andcentrally (5H).

FIG. 6 shows a schematic diagram of laminin-1 irregularityquantification.

FIG. 7 shows immunohistochemical labeling for connexins 26 and 43 incontrol cultures (7A) and following three treatments with anti-connexin43 oligodeoxynucleotides over a 24 hour period (7B), and afterconnexin31.1 specific antisense treatment (7C, 7D).

FIG. 8 shows spinal cord segments from P7 rat pups 24 hours afterplacing into culture. The control segment (1) is swollen (arrows) withtissue extruding from cut ends. Dotted lines mark the originalexcisions. Histological examination shows that cells are vacuolated andedemic. By day 5 these segments have activated microglia throughout andfew surviving neurons. In contrast, the antisense treated segment (2)has significantly reduced swelling compared to controls (p<0.001) withminimal cellular edema and vacuolation. Even after 20 days in culture,neurons in the grey matter remain viable with activated microgliarestricted to the outer edges.

FIG. 9 shows neurons from a control treated segment (9A) and aConnexin43 antisense treated segment (9B). Neurons in a control segmentare vacuolated and edemitous and the surrounding tissue is disrupted,but neurons in the treated segment appear healthy and viable.

FIG. 10 illustrates MAP-2 immunolabeling near the ends of culturedspinal cord segments five days after placing into culture. Controlsegments have few viable neurons and little MAP-2 labeling (16% show anyMAP-2 label) (10A) while 66% of treated segments have areas of MAP-2expression at the cuts ends exposed to the medium and/or adjacent toremaining white matter material.

FIG. 11 illustrates that deoxyribozymes selectively cleave specificregions of target connexin-43 mRNA in vitro. A 2.4 kb rat connexin-43mRNA (11A) and 1.2 kb mouse connexin-43 mRNA (11B) were transcribed invitro from plasmid and incubated with various deoxyribozymes for 1 hour.Region 896-953 of rat mRNA (11A) was inconclusive because nodeoxyribozymes were designed for corresponding region in mouse.Deoxyribozymes cleavage of region 367-466 in mouse mRNA (11B), does notmatch results from rat connexin-43 mRNA, probably due to the presence of200 base pair of 5′ untranslated region in rat mRNA. Defective controldeoxyribozymes with single point mutation, df605 and df783, showed thatsuch cleavages were specific. Some non-specific miss priming bydeoxyribozymes against mouse mRNA were also observed by mouse dz1007 anddz1028. Overall, deoxyribozymes targeting the 526-622, 783-885, and1007-1076 base regions showed significantly cleavage in both rat andmouse mRNA species.

FIG. 12 illustrates that deoxyribozymes selectively cleave specificregions of target connexin-26 mRNA in vitro. A 0.7 kb rat (12A) andmouse (12B) connexin-43 mRNA was transcribed in vitro from plasmid andincubated with various deoxyribozymes for 1 hour. The cleavage resultsshow that rodent connexin26 mRNA has at least two regions that aretargeted by deoxyribozymes, in the 318-379 and 493-567 base regions.Defective control deoxyribozymes with single point mutation, df351 anddf379, showed that such cleavages were specific.

FIG. 13 shows antisense oligomer penetration and stability in culturedcorneas for up to one hour. Cy3 labeled oligomers show punctate nuclearand cytoplasmic labeling one hour after delivery with Pluronic gel(13A). The rate of visible Cy3 penetration was 10-15 μm after one hourin corneal epithelium (13B). Taqman labeled oligomer probes was used tomeasure the stability of antisense oligomers inside epithelial cellsusing Lambda scan with each panel showing a 5 nm light emission spectrumtowards the red colour (13B). Intact Taqman probe shows FluorescenceResonance Energy Transfer with the red fluorescence light of TAMRArepresented on gray-scale (13D) while breakdown products are representedas green fluorescence as expected from FAM (also shown on gray-scale(13C)). The effective antisense oligomer concentration in cells could belower than that can be detected by fluorescence technique.

FIG. 14 illustrates the effects of different antisense oligomers onconnexin-43 (light, gray-scale) and connexin-26 (dark, gray-scale)protein expression were shown by in vitro deoxyribozyme mRNA cleavage.FIG. 4A shows the normal connexin-43 protein expression (light,gray-scale) in the basal cells and connexin-26 protein expression (dark,gray-scale) in the basal to intermediate cells of a Pluronic gel controltreated corneal epithelium. As14 (14B), as769 (14D), as892 (14F), allthree showing no deoxyribozyme cleavage in vitro) and DB1 sense control(14H) oligomers did not affect the expression of both connexins in exvivo cultures. as605 (14C), as783 (14E) and DB1 (14G) (all three showingpositive in vitro deoxyribozyme cleavage) showed only specificconnexin-43 knock down in the epithelium of treated corneas.

FIG. 15 shows that connexin43 antisense oligomers selectively reduceconnexin-43 proteins expression in rat corneas. Each spot represents asingle cornea with different treatments. The solid spot (black colored,DB1, as605, as783, as885, as953 and as1076) showed an average of 36% to85% reduction in connexin-43 expression when compared to white colouredspots (DB1 sense, as14, as769 and as892). All antisense oligomerspredicted by deoxyribozyme tertiary prediction assay to have little orno effect, showed an average of 85% to 134% of normal connexin-43expression. All experiments were normalised with the medium connexin-43density treated with DB 1 sense and as a result two negative oligomers(as769 and as892) showed greater connexin-43 density than DB1 sensecontrol treatment.

FIG. 16 illustrates a comparison of connexin43 mRNA levels in rat corneatreated with antisense or sense oligomers assessed using Real-Time PCR.The level is expressed as a percentage of Pluronic gel only treatedcornea. Three antisense ligodeoxynucleotides predicted by in vitro assayto be functional, DB1As, As605 and As783 (black bars), reducedconnexin43 mRNA expression to 46.8%, 44% and 25% of normal (gel onlyopen bar) levels respectively (** p<0.001). No reduction was seen forthe DB1 sense control oligomer (106%) (open bar DB1 sense). As769, whichdid not show any cleavage of Cx43 cRNA in the in vitro deoxyribozymetertiary prediction assay, served as a negative control (148%) (open barAs769).

FIG. 17 illustrates a comparison of connexin26 mRNA levels in rat corneatreated with antisense or sense (control) oligomers and assessed usingReal-Time PCR. The level is expressed as percentage of pluronic gel onlytreated cornea (gel only open bar). As330 and As375 reduced Cx26 mRNAexpression to 33% and 71% respectively (** p<0.001) (black bars). Noreduction was seen for Rv330 sense oligomer (109%) (Rv330 open bar).

DETAILED DESCRIPTION

The practice of the present inventions may employ various conventionaltechniques of molecular biology (including recombinant techniques),microbiology, cell biology, biochemistry, nucleic acid chemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, and include but are not limited to,by way of example only, Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989) and Molecular Cloning: A LaboratoryManual, third edition (Sambrook and Russel, 2001), jointly andindividually referred to herein as “Sambrook”; Oligonucleotide Synthesis(M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed.,1987); Handbook of Experimental Immunology (D. M. Weir & C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller & M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987, including supplements through2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); TheImmunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994);Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996);Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A.Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, and Harlow and Lane (1999) Using Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (jointly and individually referred to herein as Harlow andLane), Beaucage et al. eds., Current Protocols in Nucleic Acid ChemistryJohn Wiley & Sons, Inc., New York, 2000); and Agrawal, ed., Protocolsfor Oligonucleotides and Analogs, Synthesis and Properties Humana PressInc., New Jersey, 1993).

DEFINITIONS

Before further describing the inventions in general and in terms ofvarious nonlimiting specific embodiments, certain terms used in thecontext of the describing the invention are set forth. Unless indicatedotherwise, the following terms have the following meanings when usedherein and in the appended claims. Those terms that are not definedbelow or elsewhere in the specification shall have their art-recognizedmeaning.

“Antisense compounds” include different types of molecule that act toinhibit gene expression, translation, or function, including those thatact by sequence-specific targeting of mRNAs for therapeuticapplications.

Antisense compounds thus include, for example, the major nucleic-acidbased gene-silencing molecules such as, for example, chemically modifiedantisense oligodeoxyribonucleic acids (ODNs), ribozymes and siRNAs(Scherer, L. J. and Rossi, J. J. Nature Biotechnol. 21: 1457-1465(2003). Antisense compounds may also include antisense molecules suchas, for example, peptide nucleic acids (PNAs) (Braasch, D. A. and Corey,D. R., Biochemistry 41, 4503-4510 (2002)), morpholinophosphorodiamidates (Heasman, J., Dev. Biol., 243, 209-214 (2002),DNAzymes (Schubert, S. et al., Nucleic Acids Res. 31, 5982-5992 (2003).Chakraborti, S. and Banerjea, A. C., Mol. Ther. 7, 817-826 (2003),Santoro, S. W. and Joyce, G. F. Proc. Natl Acad. Sci. USA 94, 4262-4266(1997), and the recently developed 5′-end-mutated U1 small nuclear RNAs(Fortes, P. et al., Proc. Natl. Acad. Sci. USA 100, 8264-8269 (2003)).

The term “antisense sequences” refers to polynucleotides havingantisense compound activity and include, but are not limited to,sequences complementary or partially complementary, for example, to anRNA sequence. Antisense sequences thus include, for example, includenucleic acid sequences that bind to mRNA or portions thereof to blocktranscription of mRNA by ribosomes. Antisense methods are generally wellknown in the art. See, for example, PCT publication WO94/12633, andNielsen et al., 1991, Science 254:1497; Oligonucleotides and Analogues,A Practical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Research and Applications (1993, CRCPress.

As used herein, “messenger RNA” includes not only the sequenceinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotide sequences which form the 5′-untranslatedregion, the 3′-untranslated region, and the 5′ cap region, as well asribonucleotide sequences that form various secondary structures.Oligonucleotides may be formulated in accordance with this inventionwhich are targeted wholly or in part to any of these sequences.

In general, nucleic acids (including oligonucleotides) may be describedas “DNA-like” (i.e., having 2′-deoxy sugars and, generally, T ratherthan U bases) or “RNA-like” (i.e., having 2′-hydroxyl or 2′-modifiedsugars and, generally U rather than T bases). Nucleic acid helices canadopt more than one type of structure, most commonly the A- and B-forms.It is believed that, in general, oligonucleotides which have B-form-likestructure are “DNA-like” and those which have A-form-like structure are“RNA-like”.

The term “complementary” generally refers to the natural binding ofpolynucleotides under permissive salt and temperature conditions by basepairing. For example, the sequence “A-G-T” binds to the complementarysequence “T-C-A”. Complementarity between two single-stranded moleculesmay be “partial”, such that only some of the nucleic acids bind, or itmay be “complete”, such that total complementarity exists between thesingle stranded molecules. The degree of complementarity between nucleicacid molecules has significant effects on the efficiency and strength ofthe hybridization between them. “Hybridizable” and “complementary” areterms that are used to indicate a sufficient degree of complementaritysuch that stable and binding occurs between the DNA or RNA target andthe oligonucleotide. It is understood that an oligonucleotide need notbe 100% complementary to its target nucleic acid sequence to behybridizable, and it is also understood that the binding may betarget-specific, or may bind to other non-target molecules so long asthe non-specific binding does not significantly or undesirably thwartthe therapeutic or other objective. An oligonucleotide is used tointerfere with the normal function of the target molecule to cause aloss or diminution of activity, and it is preferred that there is asufficient degree of complementarity to avoid non-specific or unwantedbinding of the oligonucleotide to non-target sequences under conditionsin which specific binding is desired, i.e., under physiologicalconditions in the case of in vivo assays or therapeutic treatment or, inthe case of in vitro assays, under conditions in which the assays areconducted. In the context of certain embodiments of the invention,absolute complementarity is not required. Polynucleotides that havesufficient complementarity to form a duplex having a melting temperatureof greater than 20° C., 30° C., or 40° C. under physiologicalconditions, are generally preferred.

A “disorder” is any condition that would benefit from treatment with amolecule or composition of the invention, including those described orclaimed herein. This includes chronic and acute disorders or diseasesincluding those pathological conditions that predispose the mammal tothe disorder in question.

“Targeting” an oligonucleotide to a chosen nucleic acid target can be amultistep process. The process may begin with identifying a nucleic acidsequence whose function is to be modulated. This may be, for example, acellular gene (or mRNA made from the gene) whose expression isassociated with a particular disease state, or a foreign nucleic acid(RNA or DNA) from an infectious agent. The targeting process may alsoinclude determination of a site or sites within the nucleic acidsequence for the oligonucleotide interaction to occur such that thedesired effect, i.e., inhibition of protein expression, reduced proteindetection, or other modulation of activity, will result. Once a targetsite or sites have been identified, antisense compounds (e.g.,oligonucleotides) are chosen which are sufficiently or desirablycomplementary to the target, i.e., hybridize sufficiently and with anadequate or otherwise desired specificity, to give the desiredmodulation. In the present invention, targets include nucleic acidmolecules encoding one or more connexins. The targeting process may alsoinclude determination of a site or sites for the antisense interactionto occur such that the desired effect, will result. A preferredintragenic site, for example, is the region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of thegene. The translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),tand may also referred to as the “AUG codon,” the “start codon” or the“AUG start codon”. A minority of genes have a translation initiationcodon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA,5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms“translation initiation codon” and “start codon” can encompass manycodon sequences, even though the initiator amino acid in each instanceis typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.

The term “oligonucleotide” includes an oligomer or polymer of nucleotideor nucleoside monomers consisting of naturally occurring bases, sugarsand intersugar (backbone) linkages. The term “oligonucleotide” alsoincludes oligomers or polymers comprising non-naturally occurringmonomers, or portions thereof, which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of properties such as, for example, enhanced cellular uptake,increased stability in the presence of nucleases, or enhanced targetaffinity. A number of nucleotide and nucleoside modifications have beenshown to make the oligonucleotide into which they are incorporated moreresistant to nuclease digestion than the native oligodeoxynucleotide(ODN). Nuclease resistance is routinely measured by incubatingoligonucleotides with cellular extracts or isolated nuclease solutionsand measuring the extent of intact oligonucleotide remaining over time,usually by gel electrophoresis. Oligonucleotides, which have beenmodified to enhance their nuclease resistance, can survive intact for alonger time than unmodified oligonucleotides. A number of modificationshave also been shown to increase binding (affinity) of theoligonucleotide to its target. Affinity of an oligonucleotide for itstarget is routinely determined by measuring the Tm (melting temperature)of an oligonucleotide/target pair, which is the temperature at which theoligonucleotide and target dissociate. Dissociation is detectedspectrophotometrically. The greater the Tm, the greater the affinity ofthe oligonucleotide has for the target. In some cases, oligonucleotidemodifications which enhance target-binding affinity are also able toenhance nuclease resistance.

A “polynucleotide” means a plurality of nucleotides. Thus, the terms“nucleotide sequence” or “nucleic acid” or “polynucleotide” or“oligonucleotide” or “oligodeoxynucleotide” all refer to a heteropolymerof nucleotides or the sequence of these nucleotides. These phrases alsorefer to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA) or to any DNA-like orRNA-like material.

A polynucleotide that encodes a connexin, a connexin fragment, or aconnexin variant includes a polynucleotide encoding: the mature form ofthe connexin found in nature; the mature form of the connexin found innature and additional coding sequence, for example, a leader or signalsequence or a proprotein sequence; either of the foregoing andnon-coding sequences (for example, introns or non-coding sequence 5′and/or 3′ of the coding sequence for the mature form of the polypeptidefound in nature); fragments of the mature form of the connexin found innature; and variants of the mature form of the connexin found in nature.Thus, “connexin-encoding polynucleotide” and the like encompasspolynucleotides that include only a coding sequence for a desiredconnexin, fragment, or variant, as well as a polynucleotide thatincludes additional coding and/or non-coding sequences.

The terms “peptidomimetic” and “mimetic” refer to a synthetic chemicalcompound that may have substantially the same structural and functionalcharacteristics of the antisense polypeptides provided herein and thatmimic the connexin-specific inhibitory activity, at least in part and tosome degree. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger; TINS; 392 (1985); and Evans et al., J.Med. Chem. 30: 1229 (1987); Beeley N., Trends Biotechnol. 1994 June;12(6): 213-6; Kieber-Emmons T, et al.; Curr Opin Biotechnol. 1997August; 8(4): 435-41. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalent orenhanced therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity), such as a antisensepolynucleotide, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of, forexample, —CH2NH—, —CH2S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-,—CH(OH)CH2-, and —CH2SO—. The mimetic can be either entirely composed ofsynthetic, non-natural analogues of amino acids, or, is a chimericmolecule of partly natural peptide amino acids and partly non-naturalanalogs of amino acids. The mimetic can also incorporate any amount ofnatural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. For example, a mimetic composition is within the scopeof the invention if it is capable of down-regulating biologicalactivities of connexin proteins, such as, for example,gap-junction-mediated-cell-cell communication.

The term “composition” is intended to encompass a product comprising oneor more ingredients.

The terms “modulator” and “modulation” of connexin activity, as usedherein in its various forms, is intended to encompass inhibition inwhole or in part of the expression or activity of a connexin. Suchmodulators include small molecules agonists and antagonists of connexinfunction or expression, antisense molecules, ribozymes, triplexmolecules, and RNAi polynucleotides, gene therapy methods, and others.

The phrase “percent (%) identity” refers to the percentage of sequencesimilarity found in a comparison of two or more sequences. Percentidentity can be determined electronically using any suitable software.Likewise, “similarity” between two sequences (or one or more portions ofeither or both of them) is determined by comparing the sequence of onesequence to the a second sequence.

By “pharmaceutically acceptable” it is meant, for example, a carrier,diluent or excipient that is compatible with the other ingredients ofthe formulation and suitable for administration to a recipient thereof.

In general, the term “protein” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked viapeptide bonds, as occur when the carboxyl carbon atom of the carboxylicacid group bonded to the alpha-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of theamino group bonded to the alpha-carbon of an adjacent amino acid. Thesepeptide bond linkages, and the atoms comprising them (i.e., alpha-carbonatoms, carboxyl carbon atoms (and their substituent oxygen atoms), andamino nitrogen atoms (and their substituent hydrogen atoms)) form the“polypeptide backbone” of the protein. In addition, as used herein, theterm “protein” is understood to include the terms “polypeptide” and“peptide” (which, at times, may be used interchangeably herein).Similarly, protein fragments, analogs, derivatives, and variants are maybe referred to herein as “proteins,” and shall be deemed to be a“protein” unless otherwise indicated. The term “fragment” of a proteinrefers to a polypeptide comprising fewer than all of the amino acidresidues of the protein. As will be appreciated, a “fragment” of aprotein may be a form of the protein truncated at the amino terminus,the carboxy terminus, and/or internally (such as by natural splicing),and may also be variant and/or derivative. A “domain” of a protein isalso a fragment, and comprises the amino acid residues of the proteinrequired to confer biochemical activity corresponding to naturallyoccurring protein. Truncated molecules that are linear biologicalpolymers such as nucleic acid molecules or polypeptides may have one ormore of a deletion from either terminus of the molecule and/or one ormore deletions from a non-terminal region of the molecule, where suchdeletions may be deletions of from about 1-1500 contiguous nucleotide oramino acid residues, preferably about 1-500 contiguous nucleotide oramino acid residues and more preferably about 1-300 contiguousnucleotide or amino acid residues, including deletions of about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41-50, 51-74, 75-100, 101-150,151-200, 201-250 or 251-299 contiguous nucleotide or amino acidresidues.

The term “stringent conditions” refers to conditions that permithybridization between polynucleotides. Stringent conditions can bedefined by salt concentration, the concentration of organic solvent (forexample, formamide), temperature, and other conditions well known in theart. Stringency can be increased by reducing the concentration of salt,increasing the concentration of organic solvents, (for example,formamide), or raising the hybridization temperature. For example,stringent salt concentration will ordinarily be less than about 750 mMNaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCland 50 mM trisodium citrate, and most preferably less than about 250 mMNaCl and 25 mM trisodium citrate. Low stringency hybridization can beobtained in the absence of organic solvent, for example, formamide,while high stringency hybridization can be obtained in the presence ofan organic solvent (for example, at least about 35% formamide, mostpreferably at least about 50% formamide). Stringent temperatureconditions will ordinarily include temperatures of at least about 30°C., more preferably of at least about 37° C., and most preferably of atleast about 42° C. Varying additional parameters, for example,hybridization time, the concentration of detergent, for example, sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed, andare within the skill in the art. Stringent hybridization conditions mayalso be defined by conditions in a range from about 5° C. to about 20°C. or 25° C. below the melting temperature (Tm) of the target sequenceand a probe with exact or nearly exact complementarity to the target. Asused herein, the melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomeshalf-dissociated into single strands. Methods for calculating the Tm ofnucleic acids are well known in the art (see, for example, Berger andKimmel, 1987, Methods In Enzymology, Vol. 152: Guide To MolecularCloning Techniques, San Diego: Academic Press, Inc. and Sambrook et al.,(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Vols. 1-3, ColdSpring Harbor Laboratory). As indicated by standard references, a simpleestimate of the Tm value may be calculated by the equation:Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 MNaCl (see for example, Anderson and Young, “Quantitative FilterHybridization” in Nucleic Acid Hybridization (1985)). The meltingtemperature of a hybrid (and thus the conditions for stringenthybridization) is affected by various factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,and the like), and the concentration of salts and other components (forexample for example, the presence or absence of formamide, dextransulfate, polyethylene glycol). The effects of these factors are wellknown and are discussed in standard references in the art, see forexample, Sambrook, supra, and Ausubel, supra. Typically, stringenthybridization conditions are salt concentrations less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3,and temperatures at least about 30° C. for short probes (for example, 10to 50 nucleotides) and at least about 60° C. for long probes (forexample, greater than 50 nucleotides). As noted, stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide, in which case lower temperatures may be employed. In thepresent invention, the polynucleotide may be a polynucleotide whichhybridizes to the connexin mRNA under conditions of medium to highstringency such as 0.03M sodium chloride and 0.03M sodium citrate atfrom about 50 to about 60 degrees centigrade.

The term “therapeutically effective amount” means the amount of thesubject compound that will elicit a desired response, for example, abiological or medical response of a tissue, system, animal or human thatis sought, for example, by a researcher, veterinarian, medical doctor,or other clinician.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventive measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

The term “vector” refers to a nucleic acid molecule amplification,replication, and/or expression vehicle in the form of a plasmid, phage,viral, or other system (be it naturally occurring or synthetic) for thedelivery of nucleic acids to cells where the plasmid, phage, or virusmay be functional with bacterial, yeast, invertebrate, and/or mammalianhost cells. The vector may remain independent of host cell genomic DNAor may integrate in whole or in part with the genomic DNA. The vectorwill generally but need not contain all necessary elements so as to befunctional in any host cell it is compatible with. An “expressionvector” is a vector capable of directing the expression of an exogenouspolynucleotide, for example, a polynucleotide encoding a binding domainfusion protein, under appropriate conditions.

As described herein, the terms “homology and homologues” includepolynucleotides that may be a homologue of sequence in connexinpolynucleotide (e.g. mRNA). Such polynucleotides typically have at leastabout 70% homology, preferably at least about 80%, 90%, 95%, 97% or 99%homology with the relevant sequence, for example over a region of atleast about 15, 20, 30, 40, 50, 100 more contiguous nucleotides (of thehomologous sequence).

Homology may be calculated based on any method in the art. For examplethe UWGCG Package provides the BESTFIT program which can be used tocalculate homology (for example used on its default settings) (Devereuxet al. (1984) Nucleic Acids Research 12, p 387-395). The PILEUP andBLAST algorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F. (1993); J Mol Evol 36: 290-300; Altschul, S. F. et al.;(1990); J Mol Biol 215: 403-10. Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/L). This algorithm involvesfirst identifying high scoring sequence pair by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Extensions for the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence is less than about 1,preferably less than about 0.1, more preferably less than about 0.01,and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence byat least (or by no more than) about 1, 2, 5, 10, 15, 20 or moremutations (which may be substitutions, deletions or insertions). Thesemutations may be measured across any of the regions mentioned above inrelation to calculating homology. The homologous sequence typicallyhybridizes selectively to the original sequence at a level significantlyabove background. Selective hybridization is typically achieved usingconditions of medium to high stringency (for example 0.03M sodiumchloride and 0.03M sodium citrate at from about 50 degrees C. to about60 degrees C.). However, such hybridization may be carried out under anysuitable conditions known in the art (see Sambrook et al. (1989),Molecular Cloning: A Laboratory Manual). For example, if high stringencyis required, suitable conditions include 0.2×SSC at 60 degrees C. Iflower stringency is required, suitable conditions include 2×SSC at 60degrees C.

A “cell” means any living cell suitable for the desired application.Cells include eukaryotic and prokaryotic cells.

The term “gene product” refers to an RNA molecule transcribed from agene, or a polypeptide encoded by the gene or translated from the RNA.

The term “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (for example, “recombinantpolynucleotide”), to methods of using recombinant polynucleotides toproduce gene products in cells or other biological systems, or to apolypeptide (“recombinant protein”) encoded by a recombinantpolynucleotide. Thus, a “recombinant” polynucleotide is defined eitherby its method of production or its structure. In reference to its methodof production, the process refers to use of recombinant nucleic acidtechniques, for example, involving human intervention in the nucleotidesequence, typically selection or production. Alternatively, it can be apolynucleotide made by generating a sequence comprising a fusion of twoor more fragments that are not naturally contiguous to each other. Thus,for example, products made by transforming cells with any non-naturallyoccurring vector is encompassed, as are polynucleotides comprisingsequence derived using any synthetic oligonucleotide process. Similarly,a “recombinant” polypeptide is one expressed from a recombinantpolynucleotide.

A “recombinant host cell” is a cell that contains a vector, for example,a cloning vector or an expression vector, or a cell that has otherwisebeen manipulated by recombinant techniques to express a protein ofinterest.

This invention includes methods of using compounds and compositions forsite-specific modulation of gap-junction-associated protein expressionfor wound-healing, for example, well as, for example, surgically relatedwound-healing and/or tissue remodeling applications. The invention isuseful, for example, for correcting visual defects in conjunction withlaser surgery, for in vitro corneal engineering, and for direct eyetreatments where remodeling of the cornea is desired, including thosepreformed independent of or alternatively, in conjunction with, aprocedure (e.g., surgery) performed on the eye. Antisense modulation ofdirect cell-cell communication is preferably mediated by molecules thatdirectly or indirectly reduce coupling between cells in tissues. Suchmolecules include polynucleotides such as antisense deoxynucleotides,morpholino nucleotides, RNAi and deoxribozymes targeted to specificconnexin isoforms which result in reduced translation of the proteinisoform and interfere with the function of cell gap junctions.Administration of these antisense compounds results in the reduction ofgap-junction-mediated cell-cell communication at the site at whichconnexin expression is downregulated.

Connexins play important roles in gap junction-mediated cell-cellsignaling. Overexpression of connexin is associated with post surgicalscarring and post-trauma-induced tissue remodeling. According to certainembodiments of the invention, connexins represent useful targets fortreatment of adverse effects associated with corneal trauma andpost-surgical tissue remodeling; and for diseases and disorders wherelocalized disruption in direct cell-cell communication is desirable.Particularly, modulation of the expression of connexins can be usefulfor the site-specific modulation of gap-junction-associated proteinexpression for tissue remodeling/tissue engineering applications.Antisense compounds provided herein may be used for the modulation ofconnexins in association with ophthalmic procedures or surgeries suchas, for example, cataract surgery, intraocular lens surgery, cornealtransplant surgery and some types of glaucoma surgery, and otherprocedures described herein.

In certain embodiments, the modulation of the connexins can be appliedin ophthalmic disorders affecting the posterior segment, including theretina and lens. In another aspect of this invention, the modulation ofthe connexins can be applied in ophthalmic disorders affecting theanterior segment, which includes the cornea, conjunctiva and sclera. Inthe context of this invention, posterior segment disorders includemacular holes and degeneration, retinal tears, diabetic retinopathy,vitreoretinopathy and miscellaneous disorders. Also in the context ofthis invention, a disorder of the lens may include cataracts. In yetanother aspect, it is contemplated that the disorders of the cornea arerefractive disorders such as the sequelae of radial keratotomy, dry eye,viral conjunctivitis, ulcerative conjunctivitis and scar formation inwound healing, such as, for example, corneal epithelial wounds, and theconsequences of Sjogren's syndrome.

The present invention discloses antisense compounds, particularlyoligonucleotides, for use in modulating the function of nucleic acidmolecules encoding connexins, ultimately modulating the amount ofconnexins produced. This is accomplished by providing oligonucleotideswhich specifically hybridize with nucleic acids, preferably mRNA,encoding connexins.

This relationship between an antisense compound such as anoligonucleotide and its complementary nucleic acid target, to which ithybridizes, is commonly referred to as “antisense”. As described herein,“targeting” of an oligonucleotide to a chosen nucleic acid target istypically a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as an example, a cellular gene (or mRNA made from the gene)whose expression is associated with a particular disease state. In thepresent invention, the targets are nucleic acids encoding connexins; inother words, a gene encoding connexin, or mRNA expressed from theconnexin gene. mRNA which encodes connexin is presently a preferredtarget. The targeting process also includes determination of a site orsites within the nucleic acid sequence for the antisense interaction tooccur such that modulation of gene expression will result.

In the context of the invention, messenger RNA includes not only theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. Thus,oligonucleotides may be formulated in accordance with the presentinvention which are targeted wholly or in part to these associatedribonucleotides as well as to the informational ribonucleotides. Theoligonucleotide may therefore be specifically hybridizable with atranscription initiation site region, a translation initiation codonregion, a 5′ cap region, an intron/exon junction, coding sequences, atranslation termination codon region or sequences in the 5′- or3′-untranslated region. Since the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon.” A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (prokaryotes). It is alsoknown in the art that eukaryotic and prokaryotic genes may have two ormore alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding connexin, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region,” “AUG region” and “translation initiation codonregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation initiation codon. This region is apreferred target region. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon. This region is a preferred target region. The openreading frame (ORF) or “coding region,” which is known in the art torefer to the region between the translation initiation codon and thetranslation termination codon, is also a region which may be targetedeffectively. Other preferred target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene andthe 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns”, which are excised froma pre-mRNA transcript to yield one or more mature mRNA. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,exon-exon or intron-exon junctions, may also be preferred targetregions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Aberrant fusionjunctions due to rearrangements or deletions are also preferred targets.Targeting particular exons in alternatively spliced mRNAs may also bepreferred. It has also been found that introns can also be effective,and therefore preferred, target regions for antisense compoundstargeted, for example, to DNA or pre-mRNA.

In the context of this invention, an antisense polynucleotide may, forexample, hybridize to all or part of a connexin mRNA. Typically theantisense polynucleotide hybridizes to the ribosome binding region orthe coding region of the connexin mRNA. The polynucleotide may becomplementary to all of or a region of a connexin mRNA. For example, thepolynucleotide may be the exact complement of all or a part of connexinmRNA. The antisense polynucleotide may inhibit transcription and/ortranslation of the connexin. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation of the connexin gene, anddoes not inhibit transcription and/or translation of other genes. Theproduct may bind to the connexin gene or mRNA either (i) 5′ to thecoding sequence, and/or (ii) to the coding sequence, and/or (iii) 3′ tothe coding sequence. Generally the antisense polynucleotide will causethe expression of connexin mRNA and/or protein in a cell to be reduced.The antisense polynucleotide is generally antisense to the connexinmRNA. Such a polynucleotide may be capable of hybridizing to theconnexin mRNA and may inhibit the expression of connexin by interferingwith one or more aspects of connexin mRNA metabolism includingtranscription, mRNA processing, mRNA transport from the nucleus,translation or mRNA degradation. The antisense polynucleotide typicallyhybridizes to the connexin mRNA to form a duplex which can cause directinhibition of translation and/or destabilization of the mRNA. Such aduplex may be susceptible to degradation by nucleases.

Hybridization of antisense oligonucleotides with mRNA interferes withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

The overall effect of interference with mRNA function is modulation ofexpression of connexin. In the context of this invention “modulation”includes either inhibition or stimulation; i.e., either a decrease orincrease in expression. This modulation can be measured in ways whichare routine in the art, for example by Northern blot assay of mRNAexpression, or reverse transcriptase PCR, as taught in the examples ofthe instant application or by Western blot or ELISA assay of proteinexpression, or by an immunoprecipitation assay of protein expression.Effects on cell proliferation or tumor cell growth can also be measured,as taught in the examples of the instant application. Inhibition ispresently preferred.

Once the target site or sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired modulation. The antisense nucleic acids (DNA, RNA, modified,analogues, and the like) can be made using any suitable method forproducing a nucleic acid. Oligodeoxynucleotides directed to otherconnexin proteins can be selected in terms of their nucleotide sequenceby any art recognized approach, such as, for example, the computerprograms MacVector and OligoTech (from Oligos etc. Eugene, Oreg., USA).Equipment for such synthesis is available through several vendorsincluding MacVector and OligoTech (from Oligos etc. Eugene, Oreg., USA).For general methods relating to antisense polynucleotides, see AntisenseRNA and DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988)). See also, Dagle et al., Nucleic AcidsResearch, 19: 1805 (1991). For antisense therapy, see, for example,Uhlmann et al., Chem. Reviews, 90: 543-584 (1990). Typically, at least aportion of the nucleotide sequence is known for connexins in which theinhibition of expression is desired. Preferably, an antisense compoundis targeted to one or more specific connexin isotypes. Specific isotypesof connexins that may be targeted by the antisense compounds include,without limitation, 43, 37, 31.1, 26, and others described herein. It ispreferred, but not required, that the targeted connexins are human. Aconnexin may, for example, have a nucleobase sequence selected from SEQID NO:12-31. In certain embodiments, antisense compounds are targeted toat least 8 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO:12-31.

In certain other embodiments, a second antisense compound isadministered to the eye of the subject, wherein the second antisensecompound is targeted to at least about 8 nucleobases of a nucleic acidmolecule encoding a connexin having a nucleobase sequence selected fromSEQ ID NO:12-31, wherein said second antisense compound is targeted to adifferent connexin than a first antisense compound.

Connexin targets will vary depending upon the type of tissue to beengineered or remodeled and the precise sequence of the antisensepolynucleotide used in the invention will depend upon the targetconnexin protein. The connexin protein or proteins targeted by theoligonucleotides will be dependent upon the site at which downregulationis to be directed. This reflects the nonuniform make-up of gap junction(s) at different sites throughout the body in terms of connexin sub-unitcomposition. Some connexin proteins are however more ubiquitous thanothers in terms of distribution in tissue. As described herein,cornea-associated connexins such as connexin 43 are preferred in someembodiments. Therefore, in the context of the invention,oligonucleotides either alone or in combination, targeted towardsconnexin 43, 26, 37, 30 and/or 31.1 (e.g. see SEQ. ID. NOS: 1-11) whichare suitable for corneal engineering or remodeling application. In oneaspect of the invention, the oligodeoxynucleotides may be unmodifiedphosphodiester oligomers. In another aspect of the invention, thepolynucleotides may be single or double stranded.

It is also contemplated that oligonucleotides targeted at separateconnexin proteins may be used in combination (for example one, two,three, four or more different connexins may be targeted). For example,ODNs targeted to connexin 43, and one or more other members of theconnexin family (such as connexin 26, 30, 31.1, 37 and 43) can be usedin combination. It is also contemplated that individual antisensepolynucleotides may be specific to a particular connexin, or may target1, 2, 3 or more different connexins. Specific polynucleotides willgenerally target sequences in the connexin gene or mRNA which are notconserved between connexins, whereas non-specific polynucleotides willtarget conserved sequences. Thus, in certain embodiments, antisensecompounds are targeted to at least 8 nucleobases of a nucleic acidmolecule encoding human connexin 26, connexin 30, connexin 31.1, humanconnexin 37, connexin 43, wherein said antisense compound inhibits theexpression of a human connexin protein in cells associated with the eyeof said patient.

In certain embodiments, the nucleic acid molecules encoding a connexinhave a nucleobase sequence selected from SEQ. ID NO:12-31. In certainembodiments, the compositions target two or more human connexin proteinsand inhibit the expression of two or more human connexin proteins. Infurther certain embodiments, the antisense compounds are antisenseoligonucleotides. Exemplary antisense oligonucleotide to connexin 43selected include GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ ID NO: 1);GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (SEQ ID NO: 2); and GGC AAG AGACAC CAA AGA CAC TAC CAG CAT (SEQ ID NO: 3). An example of an antisenseoligonucleotide to connexin 26 has the sequence TCC TGA GCA ATA CCT AACGAA CAA ATA (SEQ ID NO: 4). Exemplary antisense oligonucleotide toconnexin 37 selected include 5′ CAT CTC CTT GGT GCT CAA CC 3′ (SEQ IDNO: 5) and 5′ CTG AAG TCG ACT TGG CTT GG 3′ (SEQ ID NO: 6). Exemplaryantisense oligonucleotide to connexin 30 selected include 5′ CTC AGA TAGTGG CCA GAA TGC 3′ (SEQ ID NO: 7) and 5′ TTG TCC AGG TGA CTC CAA GG 3′(SEQ ID NO: 8). Exemplary antisense oligonucleotide to connexin 31.1selected include 5′ CGT CCG AGC CCA GAA AGA TGA GGT C 3′(SEQ ID NO: 9);5′ AGA GGC GCA CGT GAG ACA C 3′ (SEQ ID NO: 10); and 5′ TGA AGA CAA TGAAGA TGT T 3′(SEQ ID NO: 11).

In a further embodiment, oligodeoxynucleotides selected from thefollowing sequences are particularly suitable for down-regulatingconnexin43 expression:

(SEQ ID NO: 1) 5′ GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC 3′(SEQ ID NO: 2) 5′ GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC 3′; and(SEQ ID NO: 3) 5′ GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 3′

In yet another embodiment, oligodeoxynucleotides selected from the groupfollowing sequences are particularly suitable for connexins 26, 37, 30,and 31.1:

(connexin26) (SEQ ID NO: 4) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA 3′(connexin37) (SEQ ID NO: 5) 5′ CAT CTC CTT GGT GCT CAA CC 3′(connexin37) (SEQ ID NO: 6) 5′ CTG AAG TCG ACT TGG CTT GG 3′(connexin30) (SEQ ID NO: 7) 5′ CTC AGA TAG TGG CCA GAA TGC 3′(connexin30) (SEQ ID NO: 8) 5′ TTG TCC AGG TGA CTC CAA GG 3′(connexin31.1) (SEQ ID NO: 9) 5′ CGT CCG AGC CCA GAA AGA TGA GGT C 3′(connexin31.1) (SEQ ID NO: 10) 5′ AGA GGC GCA CGT GAG ACA C 3′(connexin31.1) (SEQ ID NO: 11) 5′ TGA AGA CAA TGA AGA TGT T 3′

The antisense compounds provided herein generally comprise from about 8to about 40 nucleobases (i.e. from about 8 to about 40 linkednucleosides), and more typically those comprising from about 12 to about40 nucleobases, and even more typically about 30 nucleobases. Antisensecompounds comprising polynucleotides may be at least about 40, forexample at least about 60 or at least about 80, nucleotides in lengthand up to 100, 200, 300, 400, 500, 1000, 2000 or 3000 or morenucleotides in length. Suitable antisense compounds include, forexample, a 30 mer ODN.

In certain embodiments, antisense compounds are targeted to at leastabout 8 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO:12-31. In otherembodiments, the antisense compound is targeted to at least about 10, atleast about 12, at least about 14, at least about 16, at least about 18,at least about 20, at least about 25, at least about 30, and at leastabout 35 nucleobases of a nucleic acid molecule encoding a connexinhaving a nucleobase sequence selected from SEQ ID NO:12-31. The size ofthe antisense compounds, including oligonucleotides targeted to betweenat least about 8 and 35 nucleobases of a nucleic acid molecule encodinga human connexin, may be 8 nucleobases in length or longer, between 8and 100 nucleobases, between eight and 50 nucleobases, between eight and40 nucleobases, between 10 and 50 nucleobases, between 12 and 50nucleobases, between 14 and 50 nucleobases, between 16 and 50nucleobases, between 18 and 50 nucleobases, between 20 and 50nucleobases, between 25 and 50 nucleobases, between 15 and 35nucleobases in length, and the like. Other antisense compounds of theinvention may be or smaller or larger is size, for example having morethan 100 nucleobases in length.

Antisense compounds include antisense oligonucleotides (ODN), antisensepolynucleotides, deoxyribozymes, morpholino oligonucleotides, RNAimolecules or analogs thereof, siRNA molecules or analogs thereof, PNAmolecules or analogs thereof, DNAzymes or analogs thereof,5′-end-mutated U1 small nuclear RNAs and analogs thereof.

As provided herein, the antisense compound may include the use ofoligodeoxynucleotides (ODNs). ODNs are generally about 20 nucleotides inlength and act by hybridizing to pre-mRNA and mRNA to produce asubstrate for ribonuclease H (RNase H), which specifically degrades theRNA strand of the formed RNA-DNA duplexes. If modified in a way toprevent the action of RNase H, ODNs can inhibit translation of mRNA viasteric hindrance, or inhibit splicing of pre-mRNAs. ODNs andmodifications thereof have been used to target dsDNA for the inhibitionof transcription by the formation of triple helices. ODN may be obtainedby art recognized methods of automated synthesis and it is relativelystraightforward to obtain ODNs of any sequence and to block geneexpression via antisense base pairing.

In certain aspects, the phosphodiester backbone of ODNs can be modifiedto increase their efficacy as target-specific agents for blocking geneexpression. These backbone modifications were developed to improve thestability of the ODNs and to enhance their cellular uptake. The mostwidely used modification is one in which the nonbridging oxygen isreplaced by a sulfur atom, creating phosphorothioate ODNs. At least onephosphorothioate ODN has been approved by the FDA, and several otherphosphorothioate antisense ODNs are in earlier stages of clinical trialsfor a variety of cancers and inflammatory diseases.

The mechanisms of action of ODNs with respect to blocking gene functionvary depending upon the backbone of the ODN (Branch, A. D. Hepatology24, 1517-1529 (1996); Dias, N. and Stein, C. A. Mol. Cancer Thor. 1,347-355 (2002); Stein, C. A. and Cohen, J. S., Cancer Res. 48, 2659-2668(1988); Zon, G. Ann. N.Y. Acad Sci., 616, 161-172 (1990). Net negativelycharged ODNs, such as phosphodiesters and phorphorothioates, elicitRNAse H-mediated cleavage of the target mRNA. Other backbonemodifications that do not recruit RNAse H, because of their lack ofcharge or the type of helix formed with the target RNA, can beclassified as steric hindrance ODNs. Popularly used members of thislatter group include morpholinos, U—O-methyls, 2″-O-allyls, lockednucleic acids and peptide nucleic acids (PNAs). These ODNs can blocksplicing, translation, nuclear-cytoplasmic transport and translation,among other inhibition targets.

In another aspect, modulation of the connexin expression involves theuse of ribozymes. Ribozymes are RNA molecules that act as enzymes, evenin the complete absence of proteins. They have the catalytic activity ofbreaking and/or forming covalent bonds with extraordinary specificity,thereby accelerating the spontaneous rates of targeted reactions by manyorders of magnitude.

Ribozymes bind to RNA through Watson-Crick base pairing and act todegrade target RNA by catalysing the hydrolysis of the phosphodiesterbackbone. There are several different classes of ribozymes, with the‘hammerhead’ ribozyme being the most widely studied. As its nameimplies, the hammerhead ribozyme forms a unique secondary structure whenhybridized to its target mRNA. The catalytically important residueswithin the ribozyme are flanked by target-complementary sequences thatflank the target RNA cleavage site. Cleavage by a ribozyme requiresdivalent ions, such as magnesium, and is also dependent on target RNAstructure and accessibility. Co-localizing a ribozyme with a target RNAwithin the cell through the use of localization signals greatlyincreases their silencing efficiency. The hammerhead ribozymes are shortenough to be chemically synthesized or can be transcribed from vectors,allowing for the continuous production of ribozymes within cells.

The ability of RNA to serve as a catalyst was first demonstrated for theself-splicing group I intron of Tetrahymena thermophila and the RNAmoiety of RNAse. After the discovery of these two RNA enzymes,RNA-mediated catalysis has been found associated with the self-splicinggroup II introns of yeast, fungal and plant mitochondria (as well aschloroplasts) single-stranded plant viroid and virusoid RNAs, hepatitisdelta virus and a satellite RNA from Neurospora crassa mitochondria.Ribozymes occur naturally, but can also be artificially engineered forexpression and targeting of specific sequences in cis (on the samenucleic acid strand) or trans (a noncovalently linked nucleic acid). Newbiochemical activities are being developed using in vitro selectionprotocols as well as generating new ribozyme motifs that act onsubstrates other than RNA.

The group I intron of T. thermophile was the first cis-cleaving ribozymeto be converted into a trans-reacting form, which we refer to as anintron/ribozyme, making it useful both in genomic research and as apossible therapeutic. In the trans-splicing reaction, a defective exonof a targeted mRNA can be exchanged for a correct exon that iscovalently attached to the intron/ribozyme. This occurs via a splicingreaction in which the exon attached to the intron is positioned by basepairing to the target mRNA so that it can be covalently joined to the 5″end of the target transcript in a transesterification reaction. Thisreaction has been used to trans-splice wild-type sequences into sicklecell globin transcripts and mutant p53 transcripts and replace theexpanded triplets in the 3″-UTR of protein kinase transcripts in amyotonic dystrophy allele.

The endoribonuclease RNAse P is found in organisms throughout nature.This enzyme has RNA and one or more protein components depending uponthe organism from which it is isolated. The RNA component from theEscherichia coli and Bacillus subtilis enzymes can act as asite-specific cleavage agent in the absence of the protein tradercertain salt and ionic conditions. Studies of the substrate requirementsfor human and bacterial enzymes have shown that the minimal substratesfor either enzyme resemble a segment of a transfer RNA molecule. Thisstructure can be mimicked by uniquely designed antisense RNAs, whichpair to the target RNA, and serve as substrates for RNAse P-mediated,site-specific cleavage both in the test tube and in cells. It has alsobeen shown that the antisense component can be covalently joined to theRNAse P RNA, thereby directing the enzyme only to the target RNA ofinterest. Investigators have taken advantage of this property in thedesign of antisense RNAs, which pair with target mRNAs of interest tostimulate site-specific cleavage of the target and for targetedinhibition of both herpes simplex virus and cytomegalovirus in cellculture.

A number of small plant pathogenic RNAs (viroids, satellite RNAs andvirusoids), a transcript from a N. crassa mitochondrial DNA plasmid andthe animal hepatitis delta virus undergo a self-cleavage reaction invitro in the absence of protein. The reactions require neutral pH andMg²⁺. The self-cleavage reaction is an integral part of the in vivorolling circle mechanism of replication. These self-cleaving RNAs can besubdivided into groups depending on the sequence and secondary structureformed about the cleavage site. Small ribozymes have been derived from amotif found in single-stranded plant viroid and virusoid RNAs. On thebasis of a shared secondary structure and a conserved set ofnucleotides, the term “hammerhead” has been given to one group of thisself-cleavage domain. The hammerhead ribozyme is composed of 30nucleotides. The simplicity of the hammerhead catalytic domain has madeit a popular choice in the design of trans-acting ribozymes. UsingWatson-Crick base pairing, the hammerhead ribozyme can be designed tocleave any target RNA. The requirements at the cleavage site arerelatively simple, and virtually any UH sequence motif (where H is U, Cor A) can be targeted.

A second plant-derived, self-cleavage motif, initially identified in thenegative strand of the tobacco ringspot satellite RNA, has been termedthe ‘hairpin’ or “paperclip.” The hairpin ribozymes cleave RNAsubstrates in a reversible reaction that generates 2″, Y-cyclicphosphate and 5″-hydroxT1 termini-engineered versions of this catalyticmotif also cleave and turn over multiple copies of a variety of targetsin trans. Substrate requirements for the hairpin include a GUC, withcleavage occurring immediately upstream of the G. The hairpin ribozymealso catalyzes a ligation reaction, although it is more frequently usedfor cleavage reactions.

There have been numerous applications of both hammerhead and hairpinribozymes in cells for downregulating specific cellular and viraltargets. Haseloff and Gerlach designed a hammerhead motif (Haseloff andGerlach; Nature. 1988 Aug. 18; 334(6183):585-91) that can be engineeredto cleave any target by modifying the arms that base pair with righttarget. Ramemzani et al. demonstrated that this hammerhead ribozymemotif had potential therapeutic applications in a study in which therewas a virtual complete inhibition of viral gene expression andreplication using cells engineered to express an anti-humanimmunodeficiency virus (HIV) gag ribozyme (Ramezani A. et al., Frontiersin Bioscience 7:a, 29-36; 2002).

In another aspect, modulation of the connexin expression involves theuse of catalytic DNAs (or DNAzymes). Small DNAs capable of sitespecifically cleaving RNA targets have been developed via in vitroevolution (as no known DNA enzymes occur in nature). Two differentcatalytic motifs, with different cleavage site specificities have beenidentified. The most commonly used 10-20 enzymes bind to their RNAsubstrates via Watson-Crick base pairing and site specifically cleavethe target RNA, as do the hammerhead and hairpin ribozymes, resulting in2; 3″-cyclic phosphate and 5″-OH termini. Cleavage of the target mRNAsresults in their destruction and the DNAzymes recycle and cleavemultiple substrates. Catalytic DNAs are relatively inexpensive tosynthesize and have good catalytic properties, making them usefulsubstitutes for either antisense DNA or ribozymes.

Several applications of DNAzymes in cell culture have been publishedincluding the inhibition of veg FmRNA and consequent prevention ofangiogenesis, and inhibition of expression of the bcr/abl fusiontranscript characteristic of chronic myelogenous leukemia. CatalyticDNAs can be delivered exogenously, and they can be backbone-modified toin order to optimize systemic delivery in the absence of a carrier.

In another aspect of the present invention, the modulation of theconstitutive connexin gene involves the use of oligonucleotides havingmorpholino backbone structures. Summerton, J. E. and Weller, D. D. U.S.Pat. No. 5,034,506.

In another aspect of the invention, the antisense polynucleotides may bechemically modified in order to enhance their resistance to nucleasesand increase the efficacy of cell entry. For example, mixed backboneoligonucleotides (MBOs) containing segments of phosphothioateoligodeoxynucleotides and appropriately placed segments of modifiedoligodeoxyor oligoribonucleotides may be used. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonates, phosphoramidates,phosphorodithioates, N3′P5′-phosphoramidates and oligoribonucleotidephosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates, which are non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

The antisense compounds useful in this invention may includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages. Oligonucleotides having modified backbonesinclude those that retain a phosphorus atom in the backbone and thosethat do not have a phosphorus atom in the backbone. In the context ofthis invention, modified oligonucleotides that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

The antisense compounds with modified oligonucleotide backbones usefulin this invention may include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

In one aspect, it is contemplated that modified oligonucleotidebackbones that do not include a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatom and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having morpholino linkages(formed in part from the sugar portion of a nucleoside); siloxanebackbones; sulfide, sulfoxide and sulfone backbones; formacetyl andthioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; and others having mixed N, O, Sand CH2 component parts.

In one aspect, it is contemplated that oligonucleotide mimetics, boththe sugar and the internucleoside linkage, i. e. the backbone of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an oligonucleotide mimetic thathas been shown to have excellent hybridization properties, is referredto as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backboneof an oligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Further teaching of PNA compounds can be foundin Nielsen et al. (Science, 1991, 254, 1497-1500).

In one aspect, oligonucleotides with phosphorothioate backbones andoligonucleosides with heteroatom backbones, and in particular—CH2-NH—O—CH2-, —CH-2N(CH)3-O—CH-2 [known as a methylene (methylimino)or MMI backbone], —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and—O—N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone isrepresented as —O—P—O—CH2-] are contemplated. In yet another aspect,oligonucleotides having morpholino and amide backbone structures arealso contemplated.

In another aspect, it is contemplated that the modified oligonucleotidesmay also contain one or more substituted sugar moieties. For example,oligonucleotides comprising one of the following at the 2′ position: OH;F; O—, S—, or N-alkyl, O-alkyl-O-alkyl, O—, S—, or N-alkenyl, or O—, S—or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substitutedor unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)2ON(CH3)2,O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where nand m are from 1 to about 10. Other preferred oligonucleotides maycomprise one of the following at the 2′ position: C1 to C10 lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH, 3also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al. Helv.Chim. Acta 1995, 78, 486-504) i.e. an alkoxyalkoxy group. Othermodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3).2 group, also known as 2′-DMAOE, and 2′-dimethylamino-ethoxyethoxy(2′-DMAEOE), i.e., 2′-O—CH2-O—CH2-N(CH2)2.

It is further contemplated that the modifications may include 2′-methoxy(2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F).Similar modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

In another aspect, it is contemplated that the oligonucleotides may alsoinclude nucleobase (often referred to in the art simply as “base”)modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic and natural nucleobasessuch as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-amincadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in the Concise Encyclopedia Of Polymer Science And Engineering1990, pages 858-859, Kroschwitz, J. John Wiley & Sons, those disclosedby Englisch et al. (Angewandte Chemie, International Edition 1991, 30,613-722), and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications 1993, pages 289-302, Crooke, S. T. and Lebleu,B., ed., CRC Press. Certain of these nucleobases are particularly usefulfor increasing the binding affinity of the oligomeric compounds. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications 1993, CRC Press, Boca Raton, pages 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

In another aspect, it is contemplated that the modification of theoligonucleotides involves chemically linking to the oligonucleotide oneor more moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the oligonucleotide. Such moietiesinclude but are not limited to lipid moieties such as a cholesterolmoiety (Letsinger et al., Proc. Natl. Acad. Sci. USA 1989, 86,6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett.1994, 4, 1053-1059), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci. 1992, 660, 306-309; Manoharan et al.,Bioorg. Med. Chem. Let. 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res. 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J. 1991, 10, 1111-1118; Kabanov et al., FEBS Lett. 1990, 259,327-330; Svinarchuk et al., Biochimie 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett. 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther. 1996, 277, 923-937).

Also contemplated are the use of oligonucleotides which are chimericoligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. ThisRNAse H-mediated cleavage of the RNA target is distinct from the use ofribozymes to cleave nucleic acids.

Examples of chimeric oligonucleotides include but are not limited to“gapmers,” in which three distinct regions are present, normally with acentral region flanked by two regions which are chemically equivalent toeach other but distinct from the gap. A preferred example of a gapmer isan oligonucleotide in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for RNase H and is preferablycomposed of 2′-deoxynucleotides, while the flanking portions (the 5′ and3′ “wings”) are modified to have greater affinity for the target RNAmolecule but are unable to support nuclease activity (e.g. fluoro- or2′-O-methoxyethyl-substituted). Chimeric oligonucleotides are notlimited to those with modifications on the sugar, but may also includeoligonucleosides or oligonucleotides with modified backbones, e.g., withregions of phosphorothioate (P═S) and phosphodiester (P═O) backbonelinkages or with regions of MMI and P═S backbone linkages. Otherchimeras include “wingmers,” also known in the art as “hemimers,” thatis, oligonucleotides with two distinct regions. In a preferred exampleof a wingmer, the 5′ portion of the oligonucleotide serves as asubstrate for RNase H and is preferably composed of 2′-deoxynucleotides,whereas the 3′ portion is modified in such a fashion so as to havegreater affinity for the target RNA molecule but is unable to supportnuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted),or vice-versa. In one embodiment, the oligonucleotides of the presentinvention contain a 2′-O-methoxyethyl (2′-O—CH2CH2OCH3) modification onthe sugar moiety of at least one nucleotide. This modification has beenshown to increase both affinity of the oligonucleotide for its targetand nuclease resistance of the oligonucleotide. According to theinvention, one, a plurality, or all of the nucleotide subunits of theoligonucleotides may bear a 2′-O-methoxyethyl (—O—CH2CH2OCH3)modification. Oligonucleotides comprising a plurality of nucleotidesubunits having a 2′-O-methoxyethyl modification can have such amodification on any of the nucleotide subunits within theoligonucleotide, and may be chimeric oligonucleotides. Aside from or inaddition to 21-O-methoxyethyl modifications, oligonucleotides containingother modifications which enhance antisense efficacy, potency or targetaffinity are also contemplated.

The present invention also provides polynucleotides (for example, DNA,RNA, PNA or the like) that bind to double-stranded or duplex connexinnucleic acids (for example, in a folded region of the connexin RNA or inthe connexin gene), forming a triple helix-containing, or “triplex”nucleic acid. Triple helix formation results in inhibition of connexinexpression by, for example, preventing transcription of the connexingene, thus reducing or eliminating connexin activity in a cell. Withoutintending to be bound by any particular mechanism, it is believed thattriple helix pairing compromises the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules to occur.

Triplex oligo- and polynucleotides are constructed using thebase-pairing rules of triple helix formation (see, for example, Cheng etal., J. Biol. Chem. 263: 15110 (1988); Ferrin and Camerini-Otero,Science 354:1494 (1991); Ramdas et al., J. Biol. Chem. 264:17395 (1989);Strobel et al., Science 254:1639 (1991); and Rigas et al., Proc. Natl.Acad. Sci. U.S.A. 83: 9591 (1986)) and the connexin mRNA and/or genesequence. Typically, the triplex-forming oligonucleotides comprise aspecific sequence of from about 10 to about 25 nucleotides or longer“complementary” to a specific sequence in the connexin RNA or gene(i.e., large enough to form a stable triple helix, but small enough,depending on the mode of delivery, to administer in vivo, if desired).In this context, “complementary” means able to form a stable triplehelix. In one embodiment, oligonucleotides are designed to bindspecifically to the regulatory regions of the connexin gene (forexample, the connexin 5′-flanking sequence, promoters, and enhancers) orto the transcription initiation site, (for example, between −10 and +10from the transcription initiation site). For a review of recenttherapeutic advances using triplex DNA, see Gee et al., in Huber andCarr, 1994, Molecular and Immunologic Approaches, Futura Publishing Co,Mt Kisco N.Y. and Rininsland et al., 1997, Proc. Natl. Acad. Sci. USA94:5854.

The present invention also provides ribozymes useful for inhibition ofconnexin activity. The ribozymes bind and specifically cleave andinactivate connexin mRNA. Useful ribozymes can comprise 5′- and3′-terminal sequences complementary to the connexin mRNA and can beengineered by one of skill on the basis of the connexin mRNA sequence.It is contemplated that ribozymes provided herein include those havingcharacteristics of group I intron ribozymes (Cech, Biotechnology 13:323(1995)) and others of hammerhead ribozymes (Edgington, Biotechnology10:256 (1992)).

Ribozymes include those having cleavage sites such as GUA, GUU and GUC.Short RNA oligonucleotides between 15 and 20 ribonucleotides in lengthcorresponding to the region of the target connexin gene containing thecleavage site can be evaluated for secondary structural features thatmay render the oligonucleotide more desirable. The suitability ofcleavage sites may also be evaluated by testing accessibility tohybridization with complementary oligonucleotides using ribonucleaseprotection assays, or by testing for in vitro ribozyme activity inaccordance with standard procedures known in the art.

Further contemplated are antisense compounds in which antisense andribozyme functions can be combined in a single oligonucleotide.Moreover, ribozymes can comprise one or more modified nucleotides ormodified linkages between nucleotides, as described above in conjunctionwith the description of illustrative antisense oligonucleotides providedherein.

The present invention also provides polynucleotides useful forinhibition of connexin activity by methods such as RNA interference(RNAi) This and other techniques of gene suppression are well known inthe art. A review of this technique is found in Science 288:1370-1372(2000). RNAi operates on a post-transcriptional level and is sequencespecific. The process comprises introduction of RNA with partial orfully double-stranded character, or precursors of or able to encode suchRNA into the cell or into the extracellular environment.

As described by Fire et al., U.S. Pat. No. 6,506,559, the RNA maycomprise one or more strands of polymerized ribonucleotide. Thedouble-stranded structure may be formed by a single self-complementaryRNA strand or two complementary RNA strands. The RNA may includemodifications to either the phosphate-sugar backbone or the nucleosides.RNA duplex formation may be initiated either inside or outside the cell.

Studies have demonstrated that one or more ribonucleases specificallybind to and cleave double-stranded RNA into short fragments. Theribonuclease(s) remains associated with these fragments, which in turnspecifically bind to complementary mRNA, i.e., specifically bind to thetranscribed mRNA strand for the connexin gene. The mRNA for the connexingene is also degraded by the ribonuclease(s) into short fragments,thereby obviating translation and expression of the connexin gene, andso inhibiting connexin activity. Additionally, an RNA polymerase may actto facilitate the synthesis of numerous copies of the short fragments,which exponentially increases the efficiency of the system. A uniquefeature of this gene suppression pathway is that silencing is notlimited to the cells where it is initiated. The gene-silencing effectsmay be disseminated to other parts of an organism and even transmittedthrough the germ line to several generations.

In one aspect, the double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing strategy of RNAi involves the use of shortinterfering RNAs (siRNA). The use of the general RNAi approach issubject to certain limitations, including the nonspecific antiviraldefense mechanism in mammalian cells activated in response to long dsRNAmolecules (Gil J, Esteban M, “Induction of apoptosis by thedsRNA-dependent protein kinase (PKR): Mechanisms of action”. Apoptosis2000, 5:107-114). Advances in the field have been made with thedemonstration that synthetic duplexes of 21 nucleotide RNAs couldmediate gene specific RNAi in mammalian cells without invoking genericantiviral defense mechanisms (Elbashir S, et al., “Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammaliancells”. Nature 2001, 411:494-498; Caplen N. et al., Proc Natl Acad Sci2001, 98:9742-9747). Thus, siRNAs are increasingly being recognized aspowerful tools for gene-specific modulation.

As described herein, RNAi includes to a group of related gene-silencingmechanisms sharing many common biochemical components in which theterminal effector molecule is for example, but not limited to, a small21-23-nucleotide antisense RNA. One mechanism uses a relatively long,dsRNA ‘trigger; which is processed by the cellular enzyme Dicer intoshort, for example, but not limited to, 21-23-nucleotide dsRNAs,referred to as siRNAs. The strand of the siRNA complementary to thetarget RNA becomes incorporated into a multi-protein complex termed theRNA-induced silencing complex (RISC), where it serves as a guide forendonucleolytic cleavage of the mRNA strand within the target site. Thisleads to degradation of the entire mRNA; the antisense siRNA can then berecycled. In lower organisms, RNA-dependent RNA polymerase also uses theannealed guide siRNA as a primer, generating more dsRNA front thetarget, which serves in turn as a Dicer substrate, generating moresiRNAs and amplifying the siRNA signal. This pathway is commonly used asa viral defense mechanism in plants.

As described herein, the siRNA may consist of two separate, annealedsingle strands of for example, but not limited to, 21-23 nucleotides,where the terminal two 3″-nucleotides are unpaired (3″ overhang).Alternatively, the siRNA may be in the form of a single stem-loop, oftenreferred to as a short hairpin RNA (shRNA). Typically, but not always,the antisense strand of shRNAs is also completely complementary to thesense partner strand of the si/shRNA.

In mammalian cells, long dsRNAs (usually greater than 30 nucleotides inlength) trigger the interferon pathway, activating protein kinase R and2; 5″-oligoadenylate synthetase. Activation of the interferon pathwaycan lead to global downregulation of translation as well as global RNAdegradation. However, shorter siRNAs exogenously introduced intomammalian cells have been reported to bypass the interferon pathway.

The siRNA antisense product can also be derived from endogenousmicroRNAs. In human cells, regardless of the initial form (siRNAs andmicroRNAs) or processing pathway, a final mature for example, but notlimited to, 21-23-nucleotide antisense RNA that is completely homologousto the mRNA will direct mRNA cleavage. In general, the effect ofmismatches between siRNAs and target sites can vary from almost none tocomplete abrogation of activity, for reasons that are only partiallyunderstood; however, in at least one case, partial homology resulted inmRNA translation inhibition. In general, siRNA with target mismatchesdesigned to mimic a prototypical microRNA-target interaction can mediatevarying degrees of translational repression, depending on both thespecific interaction and the number of target sites in the mRNA. RNAican be activated by either exogenous delivery of preformed siRNAs or viapromoter-based expression of siRNAs or shRNAs.

Short interfering RNAs (siRNA) can be chemically synthesized orgenerated by DNA-based vectors systems. In general, this involvestranscription of short hairpin (sh)RNAs that are efficiently processedto form siRNAs within cells (Paddison P, Caudy A, Hannon G: Stablesuppression of gene expression by RNAi in mammalian cells. Proc NatlAcad Sci U.S.A 2002, 99:1443-1448; Paddison P, Caudy A, Bernstein E,Hannon G, Conklin D: Short hairpin RNAs (shRNAs) inducesequence-specific silencing in mammalian cells. Genes & Dev 2002,16:948-958; Sui G, et al., Proc Natl Acad Sci 2002, 8:5515-5520;Brummelkamp T, et al., Science 2002, 296:550-553). Therefore, in thecontext, siRNAs can be employed as an effective strategy for thetissue-specific targeting and modulation of gene expression.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors known in the art. Any other means for such synthesis may also beemployed; the actual synthesis of the oligonucleotides is wellrecognized in the art. It is well known to use similar techniques toprepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or21-alkoxyalkoxy derivatives, including 2′-O-methoxyethyloligonucleotides (Martin, P. Helv. Chim. Acta 1995, 78, 486-504). It isalso well known to use similar techniques and commercially availablemodified amidites and controlled-pore glass (CPG) products such asbiotin, fluorescein, acridine or psoralen-modified amidites and/or CPG(available from Glen Research, Sterling, Va.) to synthesizefluorescently labeled, biotinylated or other conjugatedoligonucleotides.

METHODS

In another aspect, the invention includes methods of treating a subject(e.g. patient) by administering antisense compounds to the subject.Generally, these methods include methods for tissue engineering andmethods for reducing tissue damage associated with medical procedures,including but not limited to ophthalmic procedures.

The method may comprise, for example, administering an antisensecompound to the eye of said subject in an amount sufficient to inhibitthe expression of a human connexin protein in the eye or in cellsassociated with the eye of the subject. While it is preferred that theexpression of human connexin protein is inhibited, it is envisioned thatother proteins may be targets for modulation by the antisense compounds,either alone of in combination with antisense compounds which inhibitthe expression of human connexins.

In certain embodiments, the ophthalmic procedure is an ophthalmicsurgery, including but not limited to an excimer laser photorefractivekeratectomy, a cataract extraction, corneal transplant, a surgery tocorrect refraction, a radial keratotomy, a glaucoma filtration surgery,a keratoplasty, an excimer laser photorefractive keratectomy, a cornealtransplant, a surgery to correct refraction, a ocular surface neoplasmexcision, a conjunctival or amniotic membrane graft, a pterygium andpingeculae excision, a ocular plastic surgery, a lid tumour excision, areconstructive lid procedures for congenital abnormalities, an ectropianand entropian eyelid repair, a strabismus surgery (occular muscle), apenetrating eye trauma.

In certain embodiments, antisense compounds provided herein areadministered by local or topical administration. Antisense compoundsprovided herein can also be administered, for example, systemically orby intraocular injection.

Antisense compounds provided herein can be administered to a subject ata predetermined time, for example, relative to the formation of a wound,such as that occurs in an ophthalmic procedure (e.g. surgical). Forexample, antisense compounds can be administered before an ophthalmicprocedure is performed, during an ophthalmic procedure, or after anophthalmic procedure. For example, antisense compounds may beadministered to a subject within minutes or hours before or after anophthalmic procedure is performed.

In certain embodiments, an antisense compound is administered after anophthalmic procedure is performed, and for example the antisensecompound is administered within about 4 hours of the procedure, withinabout 3 hours of the procedure, and more typically within about 2 hoursof the ophthalmic procedure, or within about 1 hour of an ophthalmicprocedure. Alternatively, an antisense compound may be administeredwithin minutes of an ophthalmic procedure, for example within 5, 10, 15,20, 30, 45, minutes of an ophthalmic procedure. Antisense compounds mayalso be administered after 4 hours of an ophthalmic procedure.

In another aspect, antisense compounds provided herein may beadministered in a methods to effect tissue engineering. In theseembodiments, and some others provided herein, antisense compounds aretypically administered over a longer periods of time, for example overthe course of days, weeks, months, or even longer, and can beadministered independent of a particular procedure performed on apatient, such as one performed on an eye.

Antisense compounds provided herein may be administered in conjunctionwith a method that increases the thickness of cornea tissue in asubject, including in methods that are not associated with an ophthalmicprocedure, and in methods in which antisense compounds are administeredin association with an ophthalmic procedure (e.g. surgery). Antisensecompounds provided herein may be administered in conjunction with amethod that promotes healing or prevents tissue damage, for example incells associated with the cornea of the subject (e.g. corneal cells).Antisense compounds provided herein may be administered in conjunctionwith a method that reduces scarring in the eye of a subject.

Antisense compounds provided herein may be administered in conjunctionwith a method that reduces hazing in the eye of a subject. Antisensecompounds provided herein may be administered in conjunction with amethod that modulates hypercellularity associated with myofibroblastdifferentiation associated with a site of a laser induced lesion,preferably in the 24 hr to 48 hr post-surgery period. Antisensecompounds provided herein may be administered in conjunction with amethod that modulates stromal remodeling and reduces haze associatedwith a site of a laser-induced lesion, preferably in the 24 hr to 72 hrpost-surgery period. Antisense compounds provided herein may beadministered in conjunction with a method that increases epithelial cellmovement in the eye of a subject. Antisense compounds provided hereinmay be administered in conjunction with a method that results in anincrease in epithelial cell movement within 12 hours of administering anantisense compound to the eye of the subject. Antisense compoundsprovided herein may be administered in conjunction with a method thatresults in an increase in epithelial cell movement within 24 hours ofadministering the antisense compound to the eye of the subject.Antisense compounds provided herein may be administered in conjunctionwith a method that prevents an increase in stromal cell density.Antisense compounds provided herein may be administered in conjunctionwith a method that inhibits stromal edema associated with a site of alaser-induced lesion in the 24 hr to 72 hr post-surgery period.Antisense compounds provided herein may be administered in conjunctionwith a method that reduces epithelial hyperplasia in the 24 hr to 72 hrpost-surgery. Antisense compounds provided herein may be administered inconjunction with a method that reduces myofibroblast activation up to 1week post-surgery. Antisense compounds provided herein may beadministered in conjunction with a method that modulates celldifferentiation that modifies the extracellular matrix. Antisensecompounds provided herein may be administered in conjunction with amethod that reduces cell proliferation.

In certain embodiments, the antisense compound decreases scar formation.In certain embodiments, the antisense compound reduces inflammation. Incertain embodiments, the antisense compound promotes wound healing. Incertain preferred embodiments, the antisense compound is used inassociation with a surgical implantation procedure. In certain preferredembodiments, the antisense compound is directed to connexin 43 and isadministered to regulate epithelial basal cell division and growth. Incertain embodiments, the antisense compound is directed to connexin 31.1and is administered to regulate outer layer keratinisation.

In other embodiments, the method promotes healing or prevents tissuedamage in cells associated with the cornea of the subject. According tocertain embodiment, antisense compounds are used in methods thatincrease the thickness of cornea tissue in a subject, or in a methodthat results in the reduction of tissue damage in corneal cells of asubject, or in a method that results in the reduction of tissue damagein cells associated with the cornea of a subject, or in a methodperformed in association with an excimer laser photorefractivekeratectomy procedure in a subject, or in a method that modulateshypercellularity associated with myofibroblast differentiationassociated with a site of a laser induced lesion, preferably in the 24hr to 48 hr post-surgery period, or in a method that modulates stromalremodeling and reduces haze associated with a site of a laser-inducedlesion, preferably in the 24 hr to 72 hr post-surgery period, or in amethod that inhibits stromal edema associated with a site of a laserinduced lesion in the 24 hr to 72 hr post-surgery period, or in a methodthat reduces epithelial hyperplasia, preferably in the 24 hr to 72 hrpost-surgery, or in a method that reduces myofibroblast activation up to1 week post-surgery, or in a method that modulates cell differentiationthat modifies the extracellular matrix, or in a method that reduces cellproliferation.

In certain embodiments, the ophthalmic procedure is cataract extraction.In a other embodiments, the ophthalmic procedure is a cornealtransplant. In other embodiments, the ophthalmic surgical procedure issurgery to correct refraction. In a other embodiments, the ophthalmicprocedure is radial keratotomy. In a other embodiments, the ophthalmicprocedure is glaucoma filtration surgery. In still other embodiments,the ophthalmic procedure is keratoplasty.

In certain embodiments, the antisense compound or composition isadministered by local or topical administration. In certain embodiments,the antisense compound or composition is administered by directapplication in the surgical wound. In certain embodiments, the antisensecompound or composition is administered by intraocular injection. Incertain embodiments, the antisense compound or composition isadministered before the surgical procedure is performed. In certainembodiments, the antisense compound or composition is administeredduring the surgical procedure. In certain non-limiting embodiments, theantisense compound or composition is administered within about 15minutes before an ophthalmic procedure is performed or up to about 2hours after an ophthalmic procedure is performed. In certain otherembodiments, for example for tissue engineering, antisense compoundsprovided herein may be administered for days or even months.

In certain further embodiments, compounds and compositions are used topromote healing or to prevent tissue damage in cells associated withcornea, where the cells associated with the cornea may be any cell inthe eye, including but not limited to corneal cells. The agents providedherein, including antisense compounds, may increase the thickness ofcornea tissue in a subject. In certain embodiments, the antisensecompound is used in combination with another compound useful forreducing tissue damage or promoting healing. For example, the antisensecompounds may be coadministered with a growth factor, cytokine, or thelike, including but not limited to FGF, NGF, NT3, PDGF, TGF, VEGF, BDGF,EGF, KGF, integrins, interleukins, plasmin, and semaphorins.

In another aspect, a pharmaceutical composition for reducing tissuedamage associated with ophthalmic surgery is provided. Thepharmaceutical composition is suitably formulated for topical or localadministration to the eye of a subject comprising an antisense compoundpresent in an amount sufficient to inhibit the expression of a humanconnexin protein in cells associated with the eye of the subject. Theantisense compound is preferably targeted to at least about 8nucleobases of a nucleic acid molecule encoding a connexin having anucleobase sequence selected from SEQ ID NO:12-31. In certainembodiments, the antisense compounds are in the form of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or vehicleand the agent or antisense compound is present in an amount effective topromote wound healing in a subject. In certain embodiments, thepharmaceutical compositions may be, for example, in a form suitable fortopical administration, including in a form suitable for topical orlocal administration to the eye of a subject. In certain furtherembodiments, the compositions and formulations may be in the form of agel, a cream, or any of the forms described herein.

In another aspect, methods of treating an injury to the central nervoussystem are provided. The method comprising administering an antisensecompound to a site proximal to a preexisting wound of the centralnervous system in association with a surgical procedure performed on apatient to treat said injury to the central nervous system, wherein saidantisense compound is targeted to at least about 8 nucleobases of anucleic acid molecule encoding a connexin having a nucleobase sequenceselected from SEQ ID NO:12-31. In certain embodiments of the method, theantisense compound is administered to reduce neuronal loss due tophysical trauma to the spinal cord. In certain embodiments of themethod, the antisense compound is administered to a site adjacent to awound that is the result of trauma. In certain embodiments of themethod, the antisense compound is administered to a site adjacent to awound that is the result of a surgery. In certain embodiments of themethod, the antisense compound is administered to a site adjacent to aspinal cord injury. In certain embodiments of the method, the antisensecompound is directed to connexin 43 and is administered to regulateepithelial basal cell division and growth. In certain embodiments of themethod, the antisense compound promotes wound healing. In certainembodiments of the method, the antisense compound reduces inflammation.In certain embodiments of the method, the antisense compound decreasesscar formation. In certain embodiments of the method, the injury to thecentral nervous system is a spinal cord injury. In certain embodimentsof the method, the antisense compound is administered to a patient atleast about 24 hours after a physical trauma to the spinal cord. Incertain embodiments of the method, the antisense compound is used inassociation with a surgical implantation procedure. In certain furtherembodiments of the method, the surgical implantation procedure isassociated with an implant pre-treated with antisense-compound topromote wound healing. In another aspect, antisense compounds capable ofpromoting the regeneration of neurons in association with a procedurefor the treatment of a preexisting wound in a patient characterized byneuronal loss are provided. In certain embodiments, the agents areantisense compounds up to 40 nucleobases in length that are targeted toat least about 8 nucleobases of a nucleic acid molecule encoding a humanconnexin and the antisense compound inhibits the expression of one ormore human connexin in association with a procedure to promote theregeneration neurons for the treatment of a preexisting wound in apatient. The wound includes those characterized by neuronal loss.Connexins that may be targeted include connexins having a nucleobasesequence selected from SEQ ID NO:12-31. In these embodiments, theantisense compounds may be administered to a patient at least 24 hoursafter a physical trauma to the spinal cord of said patient that resultedin a neuronal loss. The antisense compounds may be administered to apatient at more than 24 hours after a physical trauma to the spinal cordfor times periods of weeks, months, or years after the physical traumathat resulted in a neuronal loss.

In certain embodiments of pharmaceutical compositions and methods, theantisense compound is targeted to at least about 8 nucleobases of anucleic acid molecule encoding human connexin 30 or human connexin 37.Preferably, the antisense compound inhibits the expression of a humanconnexin 30 or 37 protein in cells associated with the eye of a patient.Other pharmaceutical compositions comprise an antisense compoundtargeted to at least about 8 nucleobases of a nucleic acid moleculeencoding a connexin (e.g. human) having a nucleobase sequence selectedfrom SEQ ID NO:12-31, and preferably the antisense compound inhibits theexpression of a human connexin in association with a procedure topromote the regeneration neurons for the treatment of a preexistingwound in a patient that is characterized by neuronal loss.

Pharmaceutical Compositions

In another aspect, the invention includes pharmaceutical compositionscomprising antisense compounds. In one embodiment, a pharmaceuticalcomposition is provided for reducing tissue damage associated with anophthalmic procedure (e.g. surgery), such that the pharmaceuticalcomposition is formulated for topical or local administration to the eyeof a subject and it comprises an antisense compound present in an amountsufficient to inhibit the expression of a human connexin protein incells associated with the eye of the subject. In certain embodiments,the antisense compound is targeted to at least about 8 nucleobases of anucleic acid molecule encoding a connexin (e.g. human) having anucleobase sequence selected from SEQ ID NO:12-31. In certainembodiments, the pharmaceutical composition includes a pharmaceuticallyacceptable carrier comprising a buffered pluronic acid or gel, forexample up to about 30% pluronic acid in phosphate buffered saline.Antisense composition may comprise different amounts of pluronic acid orgel, including without limitation in amounts up to about 5% pluronicacid in phosphate buffered saline, up to about 10% pluronic acid inphosphate buffered saline, up to about 15% pluronic acid in phosphatebuffered saline, up to about 20% pluronic acid in phosphate bufferedsaline, up to about 25% pluronic acid in phosphate buffered saline, andup to about 30% pluronic acid in phosphate buffered saline.

The antisense compounds provided herein may also include bioequivalentcompounds, including pharmaceutically acceptable salts and prodrugs.This is intended to encompass any pharmaceutically acceptable salts,esters, or salts of such esters, or any other compound which, uponadministration to an animal including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of the nucleic acids and prodrugs ofsuch nucleic acids. “Pharmaceutically acceptable salts” arephysiologically and pharmaceutically acceptable salts of the nucleicacids provided herein: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto (see, for example, Berge et al., J. ofPharma Sci. 1977, 66, 1-19).

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The oligonucleotides provided herein may additionally or alternativelybe prepared to be delivered in a “prodrug” form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the oligonucleotides maybe prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivativesaccording to the methods disclosed in WO 93/24510 to Gosselin et al.,published Dec. 9, 1993.

Antisense compounds may be formulated in a pharmaceutical composition,which may include pharmaceutically acceptable carriers, thickeners,diluents, buffers, preservatives, surface active agents, neutral orcationic lipids, lipid complexes, liposomes, penetration enhancers,carrier compounds and other pharmaceutically acceptable carriers orexcipients and the like in addition to the oligonucleotide.

Pharmaceutical compositions may also include one or more activeingredients such as interferons, antimicrobial agents, anti-inflammatoryagents, anesthetics, and the like. Formulations for parenteraladministration may include sterile aqueous solutions which may alsocontain buffers, liposomes, diluents and other suitable additives.Pharmaceutical compositions comprising the oligonucleotides providedherein may include penetration enhancers in order to enhance thealimentary delivery of the oligonucleotides. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e., fattyacids, bile salts, chelating agents, surfactants and non-surfactants(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems 1991,8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems 1990, 7, 1-33). One or more penetration enhancers from one ormore of these broad categories may be included.

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.). Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm.Pharmacol. 1992 44, 651-654).

The physiological roles of bile include the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al. McGraw-Hill, New York, N. Y., 1996, pages 934-935).Various natural bile salts, and their synthetic derivatives, act aspenetration enhancers. Thus, the term “bile salt” includes any of thenaturally occurring components of bile as well as any of their syntheticderivatives.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacids to make complex formulations. Chelating agents include, but arenot limited to, disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) [Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7, 1-33; Buuret al., J. Control Rel. 1990, 14, 43-51). Chelating agents have theadded advantage of also serving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Phamacol. 1988, 40, 252-257). Non-surfactants include, forexample, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39,621-626).

As used herein, “carrier compound” refers to a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. In contrast to a carrier compound, a“pharmaceutically acceptable carrier” (excipient) is a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal. Thepharmaceutically acceptable carrier may be liquid or solid and isselected with the planned manner of administration in mind so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.).

The compositions provided herein may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional compatible pharmaceutically-active materials such as,e.g., antipruritics, astringents, local anesthetics or anti-inflammatoryagents, or may contain additional materials useful in physicallyformulating various dosage forms of the composition of presentinvention, such as dyes, flavoring agents, preservatives, antioxidants,opacifiers, thickening agents and stabilizers. However, such materials,when added, should not unduly interfere with the biological activitiesof the components of the compositions provided herein.

Regardless of the method by which the oligonucleotides are introducedinto a patient, colloidal dispersion systems may be used as deliveryvehicles to enhance the in vivo stability of the oligonucleotides and/orto target the oligonucleotides to a particular organ, tissue or celltype. Colloidal dispersion systems include, but are not limited to,macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layers made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech. 1995, 6, 698-708).

The antisense polynucleotides may be present in a substantially isolatedform. It will be understood that the product may be mixed with carriersor diluents which will not interfere with the intended purpose of theproduct and still be regarded as substantially isolated. A product mayalso be in a substantially purified form, in which case it willgenerally comprise 90%, e.g. at least about 95%, 98% or 99% of thepolynucleotide or dry mass of the preparation.

The antisense polynucleotides may be administered topically (at the siteto be treated). Preferably the antisense polynucleotides are combinedwith a pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline. Thecomposition may be formulated for parenteral, intramuscular,intracerebral, intravenous, subcutaneous, or transdermal administration.Uptake of nucleic acids by mammalian cells is enhanced by several knowntransfection techniques, for example, those that use transfectionagents. The formulation which is administered may contain such agents.Example of these agents include cationic agents (for example calciumphosphate and DEAE-dextran) and lipofectants (for example Lipofectam™and Transfectam™).

In one aspect, the oligonucleotides may require site-specific delivery.They also require delivery over an extended period of time. Whileclearly the delivery period will be dependent upon both the site atwhich the downregulation is to be induced and the therapeutic effectwhich is desired, continuous delivery for 24 hours or longer will oftenbe required. In on aspect of the present invention, this is achieved byinclusion of the antisense compounds in a formulation together with apharmaceutically acceptable carrier or vehicle, particularly in the formof a formulation for topical administration. In particular, topicalformulations such as creams, drops, and other described herein can beemployed to regulate epithelial basal cell division and growth (usingantisense compounds targeted to connexin 43) and outer layerkeratinization (using antisense compounds targeted to connexin31.1).

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful. Compositions fororal administration include powders or granules, suspensions orsolutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids orbinders may be desirable. Compositions for parenteral administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives. In some cases it may be moreeffective to treat a patient with an oligonucleotide in conjunction withother traditional therapeutic modalities in order to increase theefficacy of a treatment regimen. As used herein, the term “treatmentregimen” is meant to encompass therapeutic, palliative and prophylacticmodalities.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC50 s found to be effective in vitroand in in vivo animal models. In general, dosage is from 0.01 mg/kg to100 mg per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 mg/kg to 100 mg perkg of body weight, once or more daily, to once every 20 years. In thetreatment or prevention of conditions which require connexin modulationan appropriate dosage level will generally be about 0.001 to 100 mg perkg patient body weight per day which can be administered in single ormultiple doses. Preferably, the dosage level will be about 1 to about 40mg/kg per day.

The oligonucleotides of this invention can be used in diagnostics,therapeutics, prophylaxis, and as research reagents and in kits. Sincethe oligonucleotides of this invention hybridize to nucleic acidsencoding connexin, sandwich, calorimetric and other assays can easily beconstructed to exploit this fact. Provision of means for detectinghybridization of oligonucleotide with the connexin genes or mRNA canroutinely be accomplished. Such provision may include enzymeconjugation, radiolabel ling or any other suitable detection systems.Kits for detecting the presence or absence of connexin may also beprepared.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

Exemplary connexins that may be targeted in certain embodimentsdescribed herein include, but are not limited to the following.

Human Cx 43, α1 (SEQ ID NO: 12)LOCUS      NM_000165   3088 bp   mRNA   linear           PRI 26-OCT-2004 DEFINITION Homo sapiens gap junction protein,           alpha 1, 43kDa (connexin 43) (GJA1),            mRNA. 1acaaaaaagc ttttacgagg tatcagcact tttctttcat tagggggaag gcgtgaggaa 61agtaccaaac agcagcggag ttttaaactt taaatagaca ggtctgagtg cctgaacttg 121ccttttcatt ttacttcatc ctccaaggag ttcaatcact tggcgtgact tcactacttt 181taagcaaaag agtggtgccc aggcaacatg ggtgactgga gcgccttagg caaactcctt 241gacaaggttc aagcctactc aactgctgga gggaaggtgt ggctgtcagt acttttcatt 301ttccgaatcc tgctgctggg gacagcggtt gagtcagcct ggggagatga gcagtctgcc 361tttcgttgta acactcagca acctggttgt gaaaatgtct gctatgacaa gtctttccca 421atctctcatg tgcgcttctg ggtcctgcag atcatatttg tgtctgtacc cacactcttg 481tacctggctc atgtgttcta tgtgatgcga aaggaagaga aactgaacaa gaaagaggaa 541gaactcaagg ttgcccaaac tgatggtgtc aatgtggaca tgcacttgaa gcagattgag 601ataaagaagt tcaagtacgg tattgaagag catggtaagg tgaaaatgcg aggggggttg 661ctgcgaacct acatcatcag tatcctcttc aagtctatct ttgaggtggc cttcttgctg 721atccagtggt acatctatgg attcagcttg agtgctgttt acacttgcaa aagagatccc 781tgcccacatc aggtggactg tttcctctct cgccccacgg agaaaaccat cttcatcatc 841ttcatgctgg tggtgtcctt ggtgtccctg gccttgaata tcattgaact cttctatgtt 901ttcttcaagg gcgttaagga tcgggttaag ggaaagagcg acccttacca tgcgaccagt 961ggtgcgctga gccctgccaa agactgtggg tctcaaaaat atgcttattt caatggctgc 1021tcctcaccaa ccgctcccct ctcgcctatg tctcctcctg ggtacaagct ggttactggc 1081gacagaaaca attcttcttg ccgcaattac aacaagcaag caagtgagca aaactgggct 1141aattacagtg cagaacaaaa tcgaatgggg caggcgggaa gcaccatctc taactcccat 1201gcacagcctt ttgatttccc cgatgataac cagaattcta aaaaactagc tgctggacat 1261gaattacagc cactagccat tgtggaccag cgaccttcaa gcagagccag cagtcgtgcc 1321agcagcagac ctcggcctga tgacctggag atctagatac aggcttgaaa gcatcaagat 1381tccactcaat tgtggagaag aaaaaaggtg ctgtagaaag tgcaccaggt gttaattttg 1441atccggtgga ggtggtactc aacagcctta ttcatgaggc ttagaaaaca caaagacatt 1501agaataccta ggttcactgg gggtgtatgg ggtagatggg tggagaggga ggggataaga 1561gaggtgcatg ttggtattta aagtagtgga ttcaaagaac ttagattata aataagagtt 1621ccattaggtg atacatagat aagggctttt tctccccgca aacaccccta agaatggttc 1681tgtgtatgtg aatgagcggg tggtaattgt ggctaaatat ttttgtttta ccaagaaact 1741gaaataattc tggccaggaa taaatacttc ctgaacatct taggtctttt caacaagaaa 1801aagacagagg attgtcctta agtccctgct aaaacattcc attgttaaaa tttgcacttt 1861gaaggtaagc tttctaggcc tgaccctcca ggtgtcaatg gacttgtgct actatatttt 1921tttattcttg gtatcagttt aaaattcaga caaggcccac agaataagat tttccatgca 1981tttgcaaata cgtatattct ttttccatcc acttgcacaa tatcattacc atcacttttt 2041catcattcct cagctactac tcacattcat ttaatggttt ctgtaaacat ttttaagaca 2101gttgggatgt cacttaacat tttttttttt tgagctaaag tcagggaatc aagccatgct 2161taatatttaa caatcactta tatgtgtgtc gaagagtttg ttttgtttgt catgtattgg 2221tacaagcaga tacagtataa actcacaaac acagatttga aaataatgca catatggtgt 2281tcaaatttga acctttctca tggatttttg tggtgtgggc caatatggtg tttacattat 2341ataattcctg ctgtggcaag taaagcacac tttttttttc tcctaaaatg tttttccctg 2401tgtatcctat tatggatact ggttttgtta attatgattc tttattttct ctcctttttt 2461taggatatag cagtaatgct attactgaaa tgaatttcct ttttctgaaa tgtaatcatt 2521gatgcttgaa tgatagaatt ttagtactgt aaacaggctt tagtcattaa tgtgagagac 2581ttagaaaaaa tgcttagagt ggactattaa atgtgcctaa atgaattttg cagtaactgg 2641tattcttggg ttttcctact taatacacag taattcagaa cttgtattct attatgagtt 2701tagcagtctt ttggagtgac cagcaacttt gatgtttgca ctaagatttt atttggaatg 2761caagagaggt tgaaagagga ttcagtagta cacatacaac taatttattt gaactatatg 2821ttgaagacat ctaccagttt ctccaaatgc cttttttaaa actcatcaca gaagattggt 2881gaaaatgctg agtatgacac ttttcttctt gcatgcatgt cagctacata aacagttttg 2941tacaatgaaa attactaatt tgtttgacat tccatgttaa actacggtca tgttcagctt 3001cattgcatgt aatgtagacc tagtccatca gatcatgtgt tctggagagt gttctttatt 3061caataaagtt ttaatttagt ataaacat Human Cx 46, α3 (SEQ ID NO: 13)LOCUS      NM_021954   1308 bp   mRNA   linear           PRI 27-OCT-2004 DEFINITION Homo sapiens gap junction protein,           alpha 3, 46kDa (connexin 46) (GJA3),            mRNA. 1atgggcgact ggagctttct gggaagactc ttagaaaatg cacaggagca ctccacggtc 61atcggcaagg tttggctgac cgtgctgttc atcttccgca tcttggtgct gggggccgcg 121gcggaggacg tgtggggcga tgagcagtca gacttcacct gcaacaccca gcagccgggc 181tgcgagaacg tctgctacga cagggccttc cccatctccc acatccgctt ctgggcgctg 241cagatcatct tcgtgtccac gcccaccctc atctacctgg gccacgtgct gcacatcgtg 301cgcatggaag agaagaagaa agagagggag gaggaggagc agctgaagag agagagcccc 361agccccaagg agccaccgca ggacaatccc tcgtcgcggg acgaccgcgg cagggtgcgc 421atggccgggg cgctgctgcg gacctacgtc ttcaacatca tcttcaagac gctgttcgag 481gtgggcttca tcgccggcca gtactttctg tacggcttcg agctgaagcc gctctaccgc 541tgcgaccgct ggccctgccc caacacggtg gactgcttca tctccaggcc cacggagaag 601accatcttca tcatcttcat gctggcggtg gcctgcgcgt ccctgctgct caacatgctg 661gagatctacc acctgggctg gaagaagctc aagcagggcg tgaccagccg cctcggcccg 721gacgcctccg aggccccgct ggggacagcc gatcccccgc ccctgccccc cagctcccgg 781ccgcccgccg ttgccatcgg gttcccaccc tactatgcgc acaccgctgc gcccctggga 841caggcccgcg ccgtgggcta ccccggggcc ccgccaccag ccgcggactt caaactgcta 901gccctgaccg aggcgcgcgg aaagggccag tccgccaagc tctacaacgg ccaccaccac 961ctgctgatga ctgagcagaa ctgggccaac caggcggccg agcggcagcc cccggcgctc 1021aaggcttacc cggcagcgtc cacgcctgca gcccccagcc ccgtcggcag cagctccccg 1081ccactcgcgc acgaggctga ggcgggcgcg gcgcccctgc tgctggatgg gagcggcagc 1141agtctggagg ggagcgccct ggcagggacc cccgaggagg aggagcaggc cgtgaccacc 1201gcggcccaga tgcaccagcc gcccttgccc ctcggagacc caggtcgggc cagcaaggcc 1261agcagggcca gcagcgggcg ggccagaccg gaggacttgg ccatctagHuman Cx 37, α4 (SEQ ID NO: 14)LOCUS      NM_002060   1601 bp   mRNA   linear           PRI 26-OCT-2004 DEFINITION Homo sapiens gap junction protein,           alpha 4, 37kDa (connexin 37) (GJA4),            mRNA. 1ctccggccat cgtccccacc tccacctggg ccgcccgcga ggcagcggac ggaggccggg 61agccatgggt gactggggct tcctggagaa gttgctggac caggtccgag agcactcgac 121cgtggtgggt aagatctggc tgacggtgct cttcatcttc cgcatcctca tcctgggcct 181ggccggcgag tcagtgtggg gtgacgagca gtcagatttc gagtgtaaca cggcccagcc 241aggctgcacc aacgtctgct atgaccaggc cttccccatc tcccacatcc gctactgggt 301gctgcagttc ctcttcgtca gcacacccac cctggtctac ctgggccatg tcatttacct 361gtctcggcga gaagagcggc tggcgcagaa ggagggggag ctgcgggcac tgccggccaa 421ggacccacag gtggagcggg cgctggccgg catagagctt cagatggcca agatctcggt 481ggcagaagat ggtcgcctgc gcattccgcg agcactgatg ggcacctatg tcgccagtgt 541gctctgcaag agtgtgctag aggcaggctt cctctatggc cagtggcgcc tgtacggctg 601gaccatggag cccgtgtttg tgtgccagcg agcaccctgc ccctacctcg tggactgctt 661tgtctctcgc cccacggaga agaccatctt catcatcttc atgttggtgg ttggactcat 721ctccctggtg cttaacctgc tggagttggt gcacctgctg tgtcgctgcc tcagccgggg 781gatgagggca cggcaaggcc aagacgcacc cccgacccag ggcacctcct cagaccctta 841cacggaccag ggtcttcttc tacctccccg tggccagggg ccctcatccc caccatgccc 901cacctacaat gggctctcat ccagtgagca gaactgggcc aacctgacca cagaggagag 961gctggcgtct tccaggcccc ctctcttcct ggacccaccc cctcagaatg gccaaaaacc 1021cccaagtcgt cccagcagct ctgcttctaa gaagcagtat gtatagaggc ctgtggctta 1081tgtcacccaa cagaggggtc ctgagaagtc tggctgcctg ggatgccccc tgccccctcc 1141tggaaggctc tgcagagatg actgggctgg ggaagcagat gcttgctggc catggagcct 1201cattgcaagt tgttcttgaa cacctgaggc cttcctgtgg cccaccaggc actacggctt 1261cctctccaga tgtgctttgc ctgagcacag acagtcagca tggaatgctc ttggccaagg 1321gtactggggc cctctggcct tttgcagctg atccagagga acccagagcc aacttacccc 1381aacctcaccc tatggaacag tcacctgtgc gcaggttgtc ctcaaaccct ctcctcacag 1441gaaaaggcgg attgaggctg ctgggtcagc cttgatcgca cagacagagc ttgtgccgga 1501tttggccctg tcaaggggac tggtgccttg ttttcatcac tccttcctag ttctactgtt 1561caagcttctg aaataaacag gacttgatca caaaaaaaaa aHuman Cx 40, α5 (SEQ ID NO: 15)LOCUS      NM_005266   2574 bp   mRNA   linear           PRI 27-OCT-2004 DEFINITION Homo sapiens gap junction protein,           alpha 5, 40kDa (connexin 40) (GJA5),           transcript variant A, mRNA. 1gcaaaaagcg tgggcagttg gagaagaagc agccagagtg tgaagaagcc cacggaagga 61aagtccaggg aggaggaaaa gaagcagaag ttttggcatc tgttccctgg ctgtgccaag 121atgggcgatt ggagcttcct gggaaatttc ctggaggaag tacacaagca ctcgaccgtg 181gtaggcaagg tctggctcac tgtcctcttc atattccgta tgctcgtgct gggcacagct 241gctgagtctt cctgggggga tgagcaggct gatttccggt gtgatacgat tcagcctggc 301tgccagaatg tctgctacga ccaggctttc cccatctccc acattcgcta ctgggtgctg 361cagatcatct tcgtctccac gccctctctg gtgtacatgg gccacgccat gcacactgtg 421cgcatgcagg agaagcgcaa gctacgggag gccgagaggg ccaaagaggt ccggggctct 481ggctcttacg agtacccggt ggcagagaag gcagaactgt cctgctggga ggaagggaat 541ggaaggattg ccctccaggg cactctgctc aacacctatg tgtgcagcat cctgatccgc 601accaccatgg aggtgggctt cattgtgggc cagtacttca tctacggaat cttcctgacc 661accctgcatg tctgccgcag gagtccctgt ccccacccgg tcaactgtta cgtatcccgg 721cccacagaga agaatgtctt cattgtcttt atgctggctg tggctgcact gtccctcctc 781cttagcctgg ctgaactcta ccacctgggc tggaagaaga tcagacagcg atttgtcaaa 841ccgcggcagc acatggctaa gtgccagctt tctggcccct ctgtgggcat agtccagagc 901tgcacaccac cccccgactt taatcagtgc ctggagaatg gccctggggg aaaattcttc 961aatcccttca gcaataatat ggcctcccaa caaaacacag acaacctggt caccgagcaa 1021gtacgaggtc aggagcagac tcctggggaa ggtttcatcc aggttcgtta tggccagaag 1081cctgaggtgc ccaatggagt ctcaccaggt caccgccttc cccatggcta tcatagtgac 1141aagcgacgtc ttagtaaggc cagcagcaag gcaaggtcag atgacctatc agtgtgaccc 1201tcctttatgg gaggatcagg accaggtggg aacaaaggag gctcagagaa gaaagacgtg 1261tcccttctga actgatgctt tctcactgtc atcactgctt ggctcctttg agccccgggt 1321ctcaatgacg ttgctcatta attctagaaa ctataaccag ggctctggga tagtaagaga 1381ggtgacaacc cacccagact gcagttccct ccccaccctc tacccagtat acgaagcctt 1441tcagattact catgaaacag ggtagaggga aagaagggaa gcatggcaaa agctggcctg 1501gaagggatag ccagagggat agaatgactc tctctctaca taccagcagc ataccaaatg 1561cgttctctaa gttcctacct ccttgacctg atcaccctcc ctcctccaag gaagagctca 1621aagttcccag ccaatagaca gcatgaatca aggaacttgc attatatgtg ctcttgaatc 1681tgttgtctcc atggaccatt cctcggagta gtggtgagat ggccttgggt tgcccttggc 1741ttctcctccc tctactcagc cttaaaaagg gcttcttgga actttaccag cagcctcagc 1801tttacaaatg ccttggtatg tacctctggc aaatgcccca ccttggtgat gttgcaacct 1861ttccttctgc tagggtgtac acctagcctg tgcaggtgtc agccctgcta gggagtcact 1921gtacacacaa actctactgg aattcctgcc aacatctgtc accctgcagc tcctttacag 1981ttcaatccaa tgatagaaac catcccttcc ctttctccct tggctgttca cccagccatt 2041ccctgaaggc cttaccaaca ggaatatcca agaagctgtt gtcccctctc gaaccctgac 2101cagatcatca gccactgagg ccagtggaat ttccccaggc cttgttaaaa caaagaaagc 2161attgtacctc tcagattccc cttgtggaaa aaaaaattct gctgtgaaga tgaaaataaa 2221aatggagaga aaacactgga aaactatttt cccctcctat ttacttcctt tgctgactgc 2281caacttagtg ccaagaggag gtgtgatgac agctatggag gcccccagat ctctctctcc 2341tggaggcttt agcaggggca aggaaatagt aggggaatct ccagctctct tggcagggcc 2401tttatttaaa gagcgcagag attcctatgt ctccctagtg cccctaatga gactgccaag 2461tgggggctgt agaaaagcct tgccttcccc agggattggc ctggtctctg tattcactgg 2521atccataatg ggttgctgtt gttttggatg aaggtaaacg atgcttggaa ttggHuman Cx 45, α7 (SEQ ID NO: 16)LOCUS      NM_005497   1191 bp   mRNA   linear           PRI 23-DEC-2003 DEFINITION Homo sapiens gap junction protein,           alpha 7, 45kDa (connexin 45) (GJA7),            mRNA. 1atgagttgga gctttctgac tcgcctgcta gaggagattc acaaccattc cacatttgtg 61gggaagatct ggctcactgt tctgattgtc ttccggatcg tccttacagc tgtaggagga 121gaatccatct attacgatga gcaaagcaaa tttgtgtgca acacagaaca gccgggctgt 181gagaatgtct gttatgatgc gtttgcacct ctctcccatg tacgcttctg ggtgttccag 241atcatcctgg tggcaactcc ctctgtgatg tacctgggct atgctatcca caagattgcc 301aaaatggagc acggtgaagc agacaagaag gcagctcgga gcaagcccta tgcaatgcgc 361tggaaacaac accgggctct ggaagaaacg gaggaggaca acgaagagga tcctatgatg 421tatccagaga tggagttaga aagtgataag gaaaataaag agcagagcca acccaaacct 481aagcatgatg gccgacgacg gattcgggaa gatgggctca tgaaaatcta tgtgctgcag 541ttgctggcaa ggaccgtgtt tgaggtgggt tttctgatag ggcagtattt tctgtatggc 601ttccaagtcc acccgtttta tgtgtgcagc agacttcctt gtcctcataa gatagactgc 661tttatttcta gacccactga aaagaccatc ttccttctga taatgtatgg tgttacaggc 721ctttgcctct tgcttaacat ttgggagatg cttcatttag ggtttgggac cattcgagac 781tcactaaaca gtaaaaggag ggaacttgag gatccgggtg cttataatta tcctttcact 841tggaatacac catctgctcc ccctggctat aacattgctg tcaaaccaga tcaaatccag 901tacaccgaac tgtccaatgc taagatcgcc tacaagcaaa acaaggccaa cacagcccag 961gaacagcagt atggcagcca tgaggagaac ctcccagctg acctggaggc tctgcagcgg 1021gagatcagga tggctcagga acgcttggat ctggcagttc aggcctacag tcaccaaaac 1081aaccctcatg gtccccggga gaagaaggcc aaagtggggt ccaaagctgg gtccaacaaa 1141agcactgcca gtagcaaatc aggggatggg aagaactctg tctggattta aHuman Cx 50, α8 (SEQ ID NO: 17)LOCUS      NM_005267   1362 bp   mRNA   linear           PRI 26-OCT-2004 DEFINITION Homo sapiens gap junction protein,           alpha 8, 50kDa (connexin 50) (GJA8),            mRNA. 1agcgccaaga gagaaagagc acatatttct ccgtgggaca ctccttgtat tggtgggtga 61gaaatgggcg actggagttt cctggggaac atcttggagg aggtgaatga gcactccacc 121gtcatcggca gagtctggct caccgtgctt ttcatcttcc ggatcctcat ccttggcacg 181gccgcagagt tcgtgtgggg ggatgagcaa tccgacttcg tgtgcaacac ccagcagcct 241ggctgcgaga acgtctgcta cgacgaggcc tttcccatct cccacattcg cctctgggtg 301ctgcagatca tcttcgtctc caccccgtcc ctgatgtacg tggggcacgc ggtgcactac 361gtccgcatgg aggagaagcg caaaagccgc gacgaggagc tgggccagca ggcggggact 421aacggcggcc cggaccaggg cagcgtcaag aagagcagcg gcagcaaagg cactaagaag 481ttccggctgg aggggaccct gctgaggacc tacatctgcc acatcatctt caagaccctc 541tttgaagtgg gcttcatcgt gggccactac ttcctgtacg ggttccggat cctgcctctg 601taccgctgca gccggtggcc ctgccccaat gtggtggact gcttcgtgtc ccggcccacg 661gagaaaacca tcttcatcct gttcatgttg tctgtggcct ctgtgtccct attcctcaac 721gtgatggagt tgagccacct gggcctgaag gggatccggt ctgccttgaa gaggcctgta 781gagcagcccc tgggggagat tcctgagaaa tccctccact ccattgctgt ctcctccatc 841cagaaagcca agggctatca gcttctagaa gaagagaaaa tcgtttccca ctatttcccc 901ttgaccgagg ttgggatggt ggagaccagc ccactgcctg ccaagccttt caatcagttc 961gaggagaaga tcagcacagg acccctgggg gacttgtccc ggggctacca agagacactg 1021ccttcctacg ctcaggtggg ggcacaagaa gtggagggcg aggggccgcc tgcagaggag 1081ggagccgaac ccgaggtggg agagaagaag gaggaagcag agaggctgac cacggaggag 1141caggagaagg tggccgtgcc agagggggag aaagtagaga cccccggagt ggataaggag 1201ggtgaaaaag aagagccgca gtcggagaag gtgtcaaagc aagggctgcc agctgagaag 1261acaccttcac tctgtccaga gctgacaaca gatgatgcca gacccctgag caggctaagc 1321aaagccagca gccgagccag gtcagacgat ctaaccgtat gaHuman Cx 36, α9, γ1 (SEQ ID NO: 18)LOCUS      NM_020660   966 bp   mRNA   linear            PRI 03-SEP-2004DEFINITION Homo sapiens connexin-36 (CX36), mRNA. 1atgggggaat ggaccatctt ggagaggctg ctagaagccg cggtgcagca gcactccact 61atgatcggaa ggatcctgtt gactgtggtg gtgatcttcc ggatcctcat tgtggccatt 121gtgggggaga cggtgtacga tgatgagcag accatgtttg tgtgcaacac cctgcagccc 181ggctgtaacc aggcctgcta tgaccgggcc ttccccatct cccacatacg ttactgggtc 241ttccagatca taatggtgtg tacccccagt ctttgcttca tcacctactc tgtgcaccag 301tccgccaagc agcgagaacg ccgctactct acagtcttcc tagccctgga cagagacccc 361cctgagtcca taggaggtcc tggaggaact gggggtgggg gcagtggtgg gggcaaacga 421gaagataaga agttgcaaaa tgctattgtg aatggggtgc tgcagaacac agagaacacc 481agtaaggaga cagagccaga ttgtttagag gttaaggagc tgactccaca cccatcaggt 541ctacgcactg catcaaaatc caagctcaga aggcaggaag gcatctcccg cttctacatt 601atccaagtgg tgttccgaaa tgccctggaa attgggttcc tggttggcca atattttctc 661tatggcttta gtgtcccagg gttgtatgag tgtaaccgct acccctgcat caaggaggtg 721gaatgttatg tgtcccggcc aactgagaag actgtctttc tagtgttcat gtttgctgta 781agtggcatct gtgttgtgct caacctggct gaactcaacc acctgggatg gcgcaagatc 841aagctggctg tgcgaggggc tcaggccaag agaaagtcaa tctatgagat tcgtaacaag 901gacctgccaa gggtcagtgt tcccaatttt ggcaggactc agtccagtga ctctgcctat 961gtgtga Human Cx 59/58, α10 (SEQ ID NO: 19)LOCUS      NM_030772   1901 bp   mRNA   linear           PRI 27-OCT-2004 DEFINITION Homo sapiens gap junction protein,           alpha 10, 59kDa (GJA10), mRNA. 1cagggagttg tggttgcaac actgtactcc agcctgggca acagagggag actctgtctc 61aacaaacaaa caaacaaaga aaaaacccca cagctatcta gggaaaaagt aaagcaacca 121gcatatagaa gtgacatatt gttatatttt caccataggt ttgctttaag aaatagtgct 181cccttcagaa tggaagaatt tatctgcctc ttatttgatg tggatcagag ctaagatggc 241tgactaaata aacatggggg actggaatct ccttggagat actctggagg aagttcacat 301ccactccacc atgattggaa agatctggct caccatcctg ttcatatttc gaatgcttgt 361tctgggtgta gcagctgaag atgtctggaa tgatgagcag tctggcttca tctgcaatac 421agaacaacca ggctgcagaa atgtatgcta cgaccaggcc tttcctatct ccctcattag 481atactgggtt ctgcaggtga tatttgtgtc ttcaccatcc ctggtctaca tgggccatgc 541attgtaccga ctgagagttc ttgaggaaga gaggcaaagg atgaaagctc agttaagagt 601agaactggag gaggtagagt ttgaaatgcc tagggatcgg aggagattgg agcaagagct 661ttgtcagctg gagaaaagga aactaaataa agctccactc agaggaacct tgctttgcac 721ttatgtgata cacattttca ctcgctctgt ggttgaagtt ggattcatga ttggacagta 781ccttttatat ggatttcact tagagccgct atttaagtgc catggccacc cgtgtccaaa 841tataatcgac tgttttgtct caagaccaac agaaaagaca atattcctat tatttatgca 901atctatagcc actatttcac ttttcttaaa cattcttgaa attttccacc taggttttaa 961aaagattaaa agagggcttt ggggaaaata caagttgaag aaggaacata atgaattcca 1021tgcaaacaag gcaaaacaaa atgtagccaa ataccagagc acatctgcaa attcactgaa 1081gcgactccct tctgcccctg attataatct gttagtggaa aagcaaacac acactgcagt 1141gtaccctagt ttaaattcat cttctgtatt ccagccaaat cctgacaatc atagtgtaaa 1201tgatgagaaa tgcattttgg atgaacagga aactgtactt tctaatgaga tttccacact 1261tagtactagt tgtagtcatt ttcaacacat cagttcaaac aataacaaag acactcataa 1321aatatttgga aaagaactta atggtaacca gttaatggaa aaaagagaaa ctgaaggcaa 1381agacagcaaa aggaactact actctagagg tcaccgttct attccaggtg ttgctataga 1441tggagagaac aacatgaggc agtcacccca aacagttttc tccttgccag ctaactgcga 1501ttggaaaccg cggtggctta gagctacatg gggttcctct acagaacatg aaaaccgggg 1561gtcacctcct aaaggtaacc tcaagggcca gttcagaaag ggcacagtca gaacccttcc 1621tccttcacaa ggagattctc aatcacttga cattccaaac actgctgatt ctttgggagg 1681gctgtccttt gagccagggt tggtcagaac ctgtaataat cctgtttgtc ctccaaatca 1741cgtagtgtcc ctaacgaaca atctcattgg taggcgggtt cccacagatc ttcagatcta 1801aacagcggtt ggcttttaga cattatatat attatcagag aagtagccta gtggtcgtgg 1861ggcacagaaa aaatagatag gggcagctct aaagaccagc tHuman Cx 46.6/47, α12 (SEQ ID NO: 20)LOCUS      AY285161   1311 bp   mRNA   linear            PRI 19-MAY-2003DEFINITION Homo sapiens connexin47 mRNA, complete            cds. 1atgagctgga gcttcctgac gcggctgctg gaggagatcc acaaccactc caccttcgtg 61ggcaaggtgt ggctcacggt gctggtggtc ttccgcatcg tgctgacggc tgtgggcggc 121gaggccatct actcggacga gcaggccaag ttcacttgca acacgcggca gccaggctgc 181gacaacgtct gctatgacgc cttcgcgccc ctgtcgcacg tgcgcttctg ggtcttccag 241attgtggtca tctccacgcc ctcggtcatg tacctgggct acgccgtgca ccgcctggcc 301cgtgcgtctg agcaggagcg gcgccgcgcc ctccgccgcc gcccggggcc acgccgcgcg 361ccccgagcgc acctgccgcc cccgcacgcc ggctggcctg agcccgccga cctgggcgag 421gaggagccca tgctgggcct gggcgaggag gaggaggagg aggagacggg ggcagccgag 481ggcgccggcg aggaagcgga ggaggcaggc gcggaggagg cgtgcactaa ggcggtcggc 541gctgacggca aggcggcagg gaccccgggc ccgaccgggc aacacgatgg gcggaggcgc 601atccagcggg agggcctgat gcgcgtgtac gtggcccagc tggtggccag ggcagctttc 661gaggtggcct tcctggtggg ccagtacctg ctgtacggct tcgaggtgcg accgttcttt 721ccctgcagcc gccagccctg cccgcacgtg gtggactgct tcgtgtcgcg ccctactgaa 781aagacggtct tcctgctggt tatgtacgtg gtcagctgcc tgtgcctgct gctcaacctc 841tgtgagatgg cccacctggg cttgggcagc gcgcaggacg cggtgcgcgg ccgccgcggc 901cccccggcct ccgcccccgc ccccgcgccg cggcccccgc cctgcgcctt ccctgcggcg 961gccgctggct tggcctgccc gcccgactac agcctggtgg tgcgggcggc cgagcgcgct 1021cgggcgcatg accagaacct ggcaaacctg gccctgcagg cgctgcgcga cggggcagcg 1081gctggggacc gcgaccggga cagttcgccg tgcgtcggcc tccctgcggc ctcccggggg 1141ccccccagag caggcgcccc cgcgtcccgg acgggcagtg ctacctctgc gggcactgtc 1201ggggagcagg gccggcccgg cacccacgag cggccaggag ccaagcccag ggctggctcc 1261gagaagggca gtgccagcag cagggacggg aagaccaccg tgtggatctg aHuman Cx 32, β1 (SEQ ID NO: 21)LOCUS      BC039198   1588 bp   mRNA   linear            PRI 07-OCT-2003DEFINITION Homo sapiens gap junction protein,           beta 1, 32kDa (connexin 32, Charcot-           Marie-Tooth neuropathy, X-linked),           mRNA (cDNA clone MGC: 22506 IMAGE:           4710239), complete cds. 1agacattctc tgggaaaggg cagcagcagc caggtgtggc agtgacaggg aggtgtgaat 61gaggcaggat gaactggaca ggtttgtaca ccttgctcag tggcgtgaac cggcattcta 121ctgccattgg ccgagtatgg ctctcggtca tcttcatctt cagaatcatg gtgctggtgg 181tggctgcaga gagtgtgtgg ggtgatgaga aatcttcctt catctgcaac acactccagc 241ctggctgcaa cagcgtttgc tatgaccaat tcttccccat ctcccatgtg cggctgtggt 301ccctgcagct catcctagtt tccaccccag ctctcctcgt ggccatgcac gtggctcacc 361agcaacacat agagaagaaa atgctacggc ttgagggcca tggggacccc ctacacctgg 421aggaggtgaa gaggcacaag gtccacatct cagggacact gtggtggacc tatgtcatca 481gcgtggtgtt ccggctgttg tttgaggccg tcttcatgta tgtcttttat ctgctctacc 541ctggctatgc catggtgcgg ctggtcaagt gcgacgtcta cccctgcccc aacacagtgg 601actgcttcgt gtcccgcccc accgagaaaa ccgtcttcac cgtcttcatg ctagctgcct 661ctggcatctg catcatcctc aatgtggccg aggtggtgta cctcatcatc cgggcctgtg 721cccgccgagc ccagcgccgc tccaatccac cttcccgcaa gggctcgggc ttcggccacc 781gcctctcacc tgaatacaag cagaatgaga tcaacaagct gctgagtgag caggatggct 841ccctgaaaga catactgcgc cgcagccctg gcaccggggc tgggctggct gaaaagagcg 901accgctgctc ggcctgctga tgccacatac caggcaacct cccatcccac ccccgaccct 961gccctgggcg agcccctcct tctcccctgc cggtgcacag gcctctgcct gctggggatt 1021actcgatcaa aaccttcctt ccctggctac ttcccttcct cccggggcct tccttttgag 1081gagctggagg ggtggggagc tagaggccac ctatgccagt gctcaaggtt actgggagtg 1141tgggctgccc ttgttgcctg cacccttccc tcttccctct ccctctctct gggaccactg 1201ggtacaagag atgggatgct ccgacagcgt ctccaattat gaaactaatc ttaaccctgt 1261gctgtcagat accctgtttc tggagtcaca tcagtgagga gggatgtggg taagaggagc 1321agagggcagg ggtgctgtgg acatgtgggt ggagaaggga gggtggccag cactagtaaa 1381ggaggaatag tgcttgctgg ccacaaggaa aaggaggagg tgtctggggt gagggagtta 1441gggagagaga agcaggcaga taagttggag caggggttgg tcaaggccac ctctgcctct 1501agtccccaag gcctctctct gcctgaaatg ttacacatta aacaggattt tacagcaaaa 1561aaaaaaaaaa aaaaaaaaaa aaaaaaaa Human Cx 26, β2 (SEQ ID NO: 22)LOCUS      NM_004004   2263 bp   mRNA   linear           PRI 28-OCT-2004 DEFINITION Homo sapiens gap junction protein,           beta 2, 26kDa (connexin 26) (GJB2),            mRNA. 1cggagcccct cggcggcgcc cggcccagga cccgcctagg agcgcaggag ccccagcgca 61gagaccccaa cgccgagacc cccgccccgg ccccgccgcg cttcctcccg acgcagagca 121aaccgcccag agtagaagat ggattggggc acgctgcaga cgatcctggg gggtgtgaac 181aaacactcca ccagcattgg aaagatctgg ctcaccgtcc tcttcatttt tcgcattatg 241atcctcgttg tggctgcaaa ggaggtgtgg ggagatgagc aggccgactt tgtctgcaac 301accctgcagc caggctgcaa gaacgtgtgc tacgatcact acttccccat ctcccacatc 361cggctatggg ccctgcagct gatcttcgtg tccacgccag cgctcctagt ggccatgcac 421gtggcctacc ggagacatga gaagaagagg aagttcatca agggggagat aaagagtgaa 481tttaaggaca tcgaggagat caaaacccag aaggtccgca tcgaaggctc cctgtggtgg 541acctacacaa gcagcatctt cttccgggtc atcttcgaag ccgccttcat gtacgtcttc 601tatgtcatgt acgacggctt ctccatgcag cggctggtga agtgcaacgc ctggccttgt 661cccaacactg tggactgctt tgtgtcccgg cccacggaga agactgtctt cacagtgttc 721atgattgcag tgtctggaat ttgcatcctg ctgaatgtca ctgaattgtg ttatttgcta 781attagatatt gttctgggaa gtcaaaaaag ccagtttaac gcattgccca gttgttagat 841taagaaatag acagcatgag agggatgagg caacccgtgc tcagctgtca aggctcagtc 901gccagcattt cccaacacaa agattctgac cttaaatgca accatttgaa acccctgtag 961gcctcaggtg aaactccaga tgccacaatg gagctctgct cccctaaagc ctcaaaacaa 1021aggcctaatt ctatgcctgt cttaattttc tttcacttaa gttagttcca ctgagacccc 1081aggctgttag gggttattgg tgtaaggtac tttcatattt taaacagagg atatcggcat 1141ttgtttcttt ctctgaggac aagagaaaaa agccaggttc cacagaggac acagagaagg 1201tttgggtgtc ctcctggggt tctttttgcc aactttcccc acgttaaagg tgaacattgg 1261ttctttcatt tgctttggaa gttttaatct ctaacagtgg acaaagttac cagtgcctta 1321aactctgtta cactttttgg aagtgaaaac tttgtagtat gataggttat tttgatgtaa 1381agatgttctg gataccatta tatgttcccc ctgtttcaga ggctcagatt gtaatatgta 1441aatggtatgt cattcgctac tatgatttaa tttgaaatat ggtcttttgg ttatgaatac 1501tttgcagcac agctgagagg ctgtctgttg tattcattgt ggtcatagca cctaacaaca 1561ttgtagcctc aatcgagtga gacagactag aagttcctag tgatggctta tgatagcaaa 1621tggcctcatg tcaaatattt agatgtaatt ttgtgtaaga aatacagact ggatgtacca 1681ccaactacta cctgtaatga caggcctgtc caacacatct cccttttcca tgactgtggt 1741agccagcatc ggaaagaacg ctgatttaaa gaggtcgctt gggaatttta ttgacacagt 1801accatttaat ggggaggaca aaatggggca ggggagggag aagtttctgt cgttaaaaac 1861agatttggaa agactggact ctaaattctg ttgattaaag atgagctttg tctacttcaa 1921aagtttgttt gcttacccct tcagcctcca attttttaag tgaaaatata actaataaca 1981tgtgaaaaga atagaagcta aggtttagat aaatattgag cagatctata ggaagattga 2041acctgaatat tgccattatg cttgacatgg tttccaaaaa atggtactcc acatacttca 2101gtgagggtaa gtattttcct gttgtcaaga atagcattgt aaaagcattt tgtaataata 2161aagaatagct ttaatgatat gcttgtaact aaaataattt tgtaatgtat caaatacatt 2221taaaacatta aaatataatc tctataataa aaaaaaaaaa aaaHuman Cx 31, β3 (SEQ ID NO: 23)LOCUS      NM_024009   2220 bp   mRNA   linear           PRI 28-OCT-2004 DEFINITION Homo sapiens gap junction protein,           beta 3, 31kDa (connexin 31) (GJB3),           transcript variant 1, mRNA. 1gaacttcttt cctggcacag gactcactgt gccccttccc gctgtgggta caaggtctgc 61cccccacccc agctctccaa agcccaccgg cctccctgga ggccgaggtc gacggcccgt 121cgcaccggga gggggggctc ccaggggtgc cccacgcacg gtcaaggtcc cgcgccaagc 181ggggaccggg ctgggccgga agcgggcacg gtactcgcgg caaactagcg tgggcgagtc 241ctgattgcag tcggacctgc cgccgcggca cttaacagtt tgcagagtgc ttcccgcccc 301tgatctcatt ggagccttcg gacagcccag cccatggcca ccgatgcccc catttcacgc 361ctgaggaagc ggaggctcag acgggccacc agcccctccg gaggctggcc cgggagcgcc 421tggcagcgtc gggtctagga gccggctccc tcctgctccc tcctccgcgc cgcccggggt 481gtgcccgccg tctgtgtgca ccactgctga gcccagctcc ggcgccctcg cctctgctgt 541gggccccggg gacgcggggt caggccaccg cgttggccag gccgctgcag gtaggcacgg 601cccccaccag gcgccatgga ctggaagaca ctccaggccc tactgagcgg tgtgaacaag 661tactccacag cgttcgggcg catctggctg tccgtggtgt tcgtcttccg ggtgctggta 721tacgtggtgg ctgcagagcg cgtgtggggg gatgagcaga aggactttga ctgcaacacc 781aagcagcccg gctgcaccaa cgtctgctac gacaactact tccccatctc caacatccgc 841ctctgggccc tgcagctcat cttcgtcaca tgcccctcgc tgctggtcat cctgcacgtg 901gcctaccgtg aggagcggga gcgccggcac cgccagaaac acggggacca gtgcgccaag 961ctgtacgaca acgcaggcaa gaagcacgga ggcctgtggt ggacctacct gttcagcctc 1021atcttcaagc tcatcattga gttcctcttc ctctacctgc tgcacactct ctggcatggc 1081ttcaatatgc cgcgcctggt gcagtgtgcc aacgtggccc cctgccccaa catcgtggac 1141tgctacattg cccgacctac cgagaagaaa atcttcacct acttcatggt gggcgcctcc 1201gccgtctgca tcgtactcac catctgtgag ctctgctacc tcatctgcca cagggtcctg 1261cgaggcctgc acaaggacaa gcctcgaggg ggttgcagcc cctcgtcctc cgccagccga 1321gcttccacct gccgctgcca ccacaagctg gtggaggctg gggaggtgga tccagaccca 1381ggcaataaca agctgcaggc ttcagcaccc aacctgaccc ccatctgacc acagggcagg 1441ggtggggcaa catgcgggct gccaatggga catgcagggc ggtgtggcag gtggagaggt 1501cctacagggg ctgagtgacc ccactctgag ttcactaagt tatgcaactt tcgttttggc 1561agatattttt tgacactggg aactgggctg tctagccggg tataggtaac ccacaggccc 1621agtgccagcc ctcaaaggac atagactttg aaacaagcga attaactatc tacgctgcct 1681gcaaggggcc acttagggca ctgctagcag ggcttcaacc aggaagggat caacccagga 1741agggatgatc aggagaggct tccctgagga cataatgtgt aagagaggtg agaagtgctc 1801ccaagcagac acaacagcag cacagaggtc tggaggccac acaaaaagtg atgctcgccc 1861tgggctagcc tcagcagacc taaggcatct ctactccetc cagaggagcc gcccagattc 1921ctgcagtgga gaggaggtct tccagcagca gcaggtctgg agggctgaga atgaacctga 1981ctagaggttc tggagatacc cagaggtccc ccaggtcatc acttggctca gtggaagccc 2041tctttcccca aatectactc cctcagcctc aggcagtggt gctcccatct tectecccac 2101aactgtgctc aggctggtgc cagcctttca gaccctgetc ccagggactt gggtggatgc 2161gctgatagaa catcctcaag acagtttcct tgaaatcaat aaatactgtg ttttataaaaHuman Cx30.3, β4 (SEQ ID NO: 24)LOCUS      NM_153212   1243 bp   mRNA   linear           PRI 27-OCT-2004 DEFINITION Homo sapiens gap junction protein,           beta 4 (connexin 30.3) (GJB4), mRNA. 1caaggctccc aaggcctgag tgggcaggta gcacccaggt atagaccttc cacgtgcagc 61acccaggaca cagccagcat gaactgggca tttctgcagg gcctgctgag tggcgtgaac 121aagtactcca cagtgctgag ccgcatctgg ctgtctgtgg tgttcatctt tcgtgtgctg 181gtgtacgtgg tggcagcgga ggaggtgtgg gacgatgagc agaaggactt tgtctgcaac 241accaagcagc ccggctgccc caacgtctgc tatgacgagt tcttccccgt gtcccacgtg 301cgcctctggg ccctacagct catcctggtc acgtgcccct cactgctcgt ggtcatgcac 361gtggcctacc gcgaggaacg cgagcgcaag caccacctga aacacgggcc caatgccccg 421tccctgtacg acaacctgag caagaagcgg ggcggactgt ggtggacgta cttgctgagc 481ctcatcttca aggccgccgt ggatgctggc ttcctctata tcttccaccg cctctacaag 541gattatgaca tgccccgcgt ggtggcctgc tccgtggagc cttgccccca cactgtggac 601tgttacatct cccggcccac ggagaagaag gtcttcacct acttcatggt gaccacagct 661gccatctgca tcctgctcaa cctcagtgaa gtcttctacc tggtgggcaa gaggtgcatg 721gagatcttcg gccccaggca ccggcggcct cggtgccggg aatgcctacc cgatacgtgc 781ccaccatatg tcctctccca gggagggcac cctgaggatg ggaactctgt cctaatgaag 841gctgggtcgg ccccagtgga tgcaggtggg tatccataac ctgcgagatc agcagataag 901atcaacaggt cccccccaca tgaggccacc caggaaaaaa ggcaggggca gtggcatcct 961tgccgtagca gggtggtgag gagggtggct gtgggggctc aggaagctcg cccaggggcc 1021aatgtgggag gttgggggta gtttggtccc tgggtcctga gcctcagggg agggaggttg 1081atagctactg gggattttgt atatggcaac agtatatgtc aaacctctta ttaaatatga 1141ttttcccagt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1201aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaHuman Cx31.1, 135 (SEQ ID NO: 25)LOCUS      NM_005268   1299 bp   mRNA   linear           PRI 23-AUG-2004 DEFINITION Homo sapiens gap junction protein,           beta 5 (connexin 31.1) (GJB5), mRNA. 1atgaaattca agctgcttgc tgagtcctat tgccggctgc tgggagccag gagagccctg 61aggagtagtc actcagtagc agctgacgcg tgggtccacc atgaactgga gtatctttga 121gggactcctg agtggggtca acaagtactc cacagccttt gggcgcatct ggctgtctct 181ggtcttcatc ttccgcgtgc tggtgtacct ggtgacggcc gagcgtgtgt ggagtgatga 241ccacaaggac ttcgactgca atactcgcca gcccggctgc tccaacgtct gctttgatga 301gttcttccct gtgtcccatg tgcgcctctg ggccctgcag cttatcctgg tgacatgccc 361ctcactgctc gtggtcatgc acgtggccta ccgggaggtt caggagaaga ggcaccgaga 421agcccatggg gagaacagtg ggcgcctcta cctgaacccc ggcaagaagc ggggtgggct 481ctggtggaca tatgtctgca gcctagtgtt caaggcgagc gtggacatcg cctttctcta 541tgtgttccac tcattctacc ccaaatatat cctccctcct gtggtcaagt gccacgcaga 601tccatgtccc aatatagtgg actgcttcat ctccaagccc tcagagaaga acattttcac 661cctcttcatg gtggccacag ctgccatctg catcctgctc aacctcgtgg agctcatcta 721cctggtgagc aagagatgcc acgagtgcct ggcagcaagg aaagctcaag ccatgtgcac 781aggtcatcac ccccacggta ccacctcttc ctgcaaacaa gacgacctcc tttcgggtga 841cctcatcttt ctgggctcag acagtcatcc tcctctctta ccagaccgcc cccgagacca 901tgtgaagaaa accatcttgt gaggggctgc ctggactggt ctggcaggtt gggcctggat 961ggggaggctc tagcatctct cataggtgca acctgagagt gggggagcta agccatgagg 1021taggggcagg caagagagag gattcagacg ctctgggagc cagttcctag tcctcaactc 1081cagccacctg ccccagctcg acggcactgg gccagttccc cctctgctct gcagctcggt 1141ttccttttct agaatggaaa tagtgagggc caatgcccag ggttggaggg aggagggcgt 1201tcatagaaga acacacatgc gggcaccttc atcgtgtgtg gcccactgtc agaacttaat 1261aaaagtcaac tcatttgctg gaaaaaaaaa aaaaaaaaaHuman Cx 30, β6 (SEQ ID NO: 26)LOCUS      BC038934   1805 bp   mRNA   linear            PRI 30-JUN-2004DEFINITION Homo sapiens gap junction protein,           beta 6 (connexin 30), mRNA (cDNA           clone MGC: 45195 IMAGE: 5196769),            complete cds. 1ctgggaagac gctggtcagt tcacctgccc cactggttgt tttttaaaca aattctgata 61caggcgacat cctcactgac cgagcaaaga ttgacattcg tatcatcact gtgcaccatt 121ggcttctagg cactccagtg gggtaggaga aggaggtctg aaaccctcgc agagggatct 181tgccctcatt ctttgggtct gaaacactgg cagtcgttgg aaacaggact cagggataaa 241ccagcgcaat ggattggggg acgctgcaca ctttcatcgg gggtgtcaac aaacactcca 301ccagcatcgg gaaggtgtgg atcacagtca tctttatttt ccgagtcatg atcctcgtgg 361tggctgccca ggaagtgtgg ggtgacgagc aagaggactt cgtctgcaac acactgcaac 421cgggatgcaa aaatgtgtgc tatgaccact ttttcccggt gtcccacatc cggctgtggg 481ccctccagct gatcttcgtc tccaccccag cgctgctggt ggccatgcat gtggcctact 541acaggcacga aaccactcgc aagttcaggc gaggagagaa gaggaatgat ttcaaagaca 601tagaggacat taaaaagcag aaggttcgga tagaggggtc gctgtggtgg acgtacacca 661gcagcatctt tttccgaatc atctttgaag cagcctttat gtatgtgttt tacttccttt 721acaatgggta ccacctgccc tgggtgttga aatgtgggat tgacccctgc cccaaccttg 781ttgactgctt tatttctagg ccaacagaga agaccgtgtt taccattttt atgatttctg 841cgtctgtgat ttgcatgctg cttaacgtgg cagagttgtg ctacctgctg ctgaaagtgt 901gttttaggag atcaaagaga gcacagacgc aaaaaaatca ccccaatcat gccctaaagg 961agagtaagca gaatgaaatg aatgagctga tttcagatag tggtcaaaat gcaatcacag 1021gtttcccaag ctaaacattt caaggtaaaa tgtagctgcg tcataaggag acttctgtct 1081tctccagaag gcaataccaa cctgaaagtt ccttctgtag cctgaagagt ttgtaaatga 1141ctttcataat aaatagacac ttgagttaac tttttgtagg atacttgctc cattcataca 1201caacgtaatc aaatatgtgg tccatctctg aaaacaagag actgcttgac aaaggagcat 1261tgcagtcact ttgacaggtt ccttttaagt ggactctctg acaaagtggg tactttctga 1321aaatttatat aactgttgtt gataaggaac atttatccag gaattgatac ttttattagg 1381aaaagatatt tttataggct tggatgtttt tagttctgac tttgaattta tataaagtat 1441ttttataatg actggtcttc cttacctgga aaaacatgcg atgttagttt tagaattaca 1501ccacaagtat ctaaatttgg aacttacaaa gggtctatct tgtaaatatt gttttgcatt 1561gtctgttggc aaatttgtga actgtcatga tacgcttaag gtggaaagtg ttcattgcac 1621aatatatttt tactgctttc tgaatgtaga cggaacagtg tggaagcaga aggctttttt 1681aactcatccg tttgccaatc attgcaaaca actgaaatgt ggatgtgatt gcctcaataa 1741agctcgtccc cattgcttaa gccttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1801aaaaa Human Cx31.9, c1 (SEQ ID NO: 27)LOCUS      NM_152219   2094 bp   mRNA   linear           PRI 23-AUG-2004 DEFINITION Homo sapiens gap junction protein,           chi 1, 31.9kDa (connexin 31.9) (GJC1),            mRNA. 1aaatgaaaga gggagcagga ggcgccggtc ccagccacct cccaaggtcc ctggctcagc 61tctgacaccc cagtcccggc cccagggtga gtggggttgg gtggcggttt aggggcacca 121ggggcgtgtg gggacctgtg taagtgtggg gtggggagga tctcaggaga tgtggaggct 181ggaggcacag gaggccaggg aggagggaga agcctggtgc cgcactccca ccacgctggg 241gtaggagggc agggacacct ccgacaaagg accctgtgag agttatgaaa gcggagttgc 301ctctgtacca gccccccacc ctgagaggag ttcactgcag taaaaatggt gagagaaatg 361gtgggccaag aaaggagtgg tctcgctgcc tctgccactc ccactcctcc catgggcacc 421aaattgggtc tagcgtctcg ggttcgaggc tccactcttc ccacagcatc cttgacagct 481aagggcaccg ctgggtttcc gcttccgaaa ccaggcaagt caggggctgg tccagctgat 541ctccaaggtc cttcctaaga atctgggatc tggaggatcc cagggtcgaa cggagacggc 601tcagggggtg cggctaaaat gcaaatgggg gatcctcccc agcacccatc ggtcccaaag 661agaaggtaac ccatagctga gcgtcgcctg ctcccctcgg gccctcccgt ggccctccgt 721ttcatactgg tctcatcgct aaacccgggc ctctcctacc tcacgactca ccctgaagtc 781agagaaggtc caacggaccc caccccgata ggcttggaag gggcaggggt ccctgacttg 841ccccatcccc tgactccccg ccccgcgtcc ccagcgccat gggggagtgg gcgttcctgg 901gctcgctgct ggacgccgtg cagctgcagt cgccgctcgt gggccgcctc tggctggtgg 961tcatgctgat cttccgcatc ctggtgctgg ccacggtggg cggcgccgtg ttcgaggacg 1021agcaagagga gttcgtgtgc aacacgctgc agccgggctg tcgccagacc tgctacgacc 1081gcgccttccc ggtctcccac taccgcttct ggctcttcca catcctgctg ctctcggcgc 1141ccccggtgct gttcgtcgtc tactccatgc accgggcagg caaggaggcg ggcggcgctg 1201aggcggcggc gcagtgcgcc cccggactgc ccgaggccca gtgcgcgccg tgcgccctgc 1261gcgcccgccg cgcgcgccgc tgctacctgc tgagcgtggc gctgcgcctg ctggccgagc 1321tgaccttcct gggcggccag gcgctgctct acggettccg cgtggccccg cacttcgcgt 1381gcgccggtcc gccctgcccg cacacggtcg actgcttcgt gagccggccc accgagaaga 1441ccgtcttcgt gctcttctat ttcgcggtgg ggctgctgtc ggcgctgctc agcgtagccg 1501agctgggcca cctgctctgg aagggccgcc cgcgcgccgg ggagcgtgac aaccgctgca 1561accgtgcaca cgaagaggcg cagaagctgc tcccgccgcc gccgccgcca cctattgttg 1621tcacttggga agaaaacaga caccttcaag gagagggctc ccctggtagc ccccacccca 1681agacagagct ggatgcccct cgcttccgta gggaaagcac ttctcctgca ggatggcatt 1741gctctctccc cttccatggc acgtagtatg tgctcagtaa atatgtgttg gatgagaaac 1801tgaaggtgtc cccaggccta caccactgcc atgcccgaac actatccatg ctatggtggg 1861caccatctct ctgatgacag ttctgtgtcc acaacccaga cccctccaca caaacccaga 1921tggggctgtg ccgctgtttt ccagatgtat tcattcaaca aatatttgta gggtacctac 1981tgtgtgtcag aagatgttca agatcagcat catccgatgg aaatagcata tgagccatgt 2041atgtagtttc aagtttttca ttagccgcat taaaaaagta aaaggaaaca aatgHuman Cx 29/31.3, e1 (SEQ ID NO: 28)LOCUS      AF503615   840 bp   mRNA   linear            PRI 07-AUG-2002DEFINITION Homo sapiens connexin 31.3 mRNA,            complete cds. 1atgtgtggca ggttcctgcg gcggctgctg gcggaggaga gccggcgctc cacccccgtg 61gggcgcctct tgcttcccgt gctcctggga ttccgccttg tgctgctggc tgccagtggg 121cctggagtct atggtgatga gcagagtgaa ttcgtgtgtc acacccagca gccgggctgc 181aaggctgcct gcttcgatgc cttccacccc ctctccccgc tgcgtttctg ggtcttccag 241gtcatcttgg tggctgtacc cagcgccctc tatatgggtt tcactctgta tcacgtgatc 301tggcactggg aattatcagg aaaggggaag gaggaggaga ccctgatcca gggacgggag 361ggcaacacag atgtcccagg ggctggaagc ctcaggctgc tctgggctta tgtggctcag 421ctgggggctc ggcttgtcct ggagggggca gccctggggt tgcagtacca cctgtatggg 481ttccagatgc ccagctcctt tgcatgtcgc cgagaacctt gccttggtag tataacctgc 541aatctgtccc gcccctctga gaagaccatt ttcctaaaga ccatgtttgg agtcagcggt 601ttctgtctct tgtttacttt tttggagctt gtgcttctgg gtttggggag atggtggagg 661acctggaagc acaaatcttc ctcttctaaa tacttcctaa cttcagagag caccagaaga 721cacaagaaag caaccgatag cctcccagtg gtggaaacca aagagcaatt tcaagaagca 781gttccaggaa gaagcttagc ccaggaaaaa caaagaccag ttggacccag agatgcctgaHuman Cx 25 (SEQ ID NO: 29) LOCUS      HSA414563   672 bp   DNA   linear           PRI 30-NOV-2001DEFINITION Homo sapiens CX25 gene for connexin25. 1atgagttgga tgttcctcag agatctcctg agtggagtaa ataaatactc cactgggact 61ggatggattt ggctggctgt cgtgtttgtc ttccgtttgc tggtctacat ggtggcagca 121gagcacatgt ggaaagatga gcagaaagag tttgagtgca acagtagaca gcccggttgc 181aaaaatgtgt gttttgatga cttcttcccc atttcccaag tcagactttg ggccttacaa 241ctgataatgg tctccacacc ttcacttctg gtggttttac atgtagccta tcatgagggt 301agagagaaaa ggcacagaaa gaaactctat gtcagcccag gtacaatgga tgggggccta 361tggtacgctt atcttatcag cctcattgtt aaaactggtt ttgaaattgg cttccttgtt 421ttattttata agctatatga tggctttagt gttccctacc ttataaagtg tgatttgaag 481ccttgtccca acactgtgga ctgcttcatc tccaaaccca ctgagaagac gatcttcatc 541ctcttcttgg tcatcacctc atgcttgtgt attgtgttga atttcattga actgagtttt 601ttggttctca agtgctttat taagtgctgt ctccaaaaat atttaaaaaa acctcaagtc 661ctcagtgtgt ga Human Cx40.1 (SEQ ID NO: 30)LOCUS      HSA414564   1113 bp   mRNA   linear           PRI 30-NOV-2001 DEFINITION Homo sapiens mRNA for connexin40.1           (CX40.1 gene). 1 atggaaggcg tggacttgct agggtttctc atcatcacattaaactgcaa cgtgaccatg 61 gtaggaaagc tctggttcgt cctcacgatg ctgctgcggatgctggtgat tgtcttggcg 121 gggcgacccg tctaccagga cgagcaggag aggtttgtctgcaacacgct gcagccggga 181 tgcgccaatg tttgctacga cgtcttctcc cccgtgtctcacctgcggtt ctggctgatc 241 cagggcgtgt gcgtcctcct cccctccgcc gtcttcagcgtctatgtcct gcaccgagga 301 gccacgctcg ccgcgctggg cccccgccgc tgccccgacccccgggagcc ggcctccggg 361 cagagacgct gcccgcggcc attcggggag cgcggcggcctccaggtgcc cgacttttcg 421 gccggctaca tcatccacct cctcctccgg accctgctggaggcagcctt cggggccttg 481 cactactttc tctttggatt cctggccccg aagaagttcccttgcacgcg ccctccgtgc 541 acgggcgtgg tggactgcta cgtgtcgcgg cccacagagaagtccctgct gatgctgttc 601 ctctgggcgg tcagcgcgct gtcttttctg ctgggcctcgccgacctggt ctgcagcctg 661 cggcggcgga tgcgcaggag gccgggaccc cccacaagcccctccatccg gaagcagagc 721 ggagcctcag gccacgcgga gggacgccgg actgacgaggagggtgggcg ggaggaagag 781 ggggcaccgg cgcccccggg tgcacgcgcc ggaggggagggggctggcag ccccaggcgt 841 acatccaggg tgtcagggca cacgaagatt ccggatgaggatgagagtga ggtgacatcc 901 tccgccagcg aaaagctggg cagacagccc cggggcaggccccaccgaga ggccgcccag 961 gaccccaggg gctcaggatc cgaggagcag ccctcagcagcccccagccg cctggccgcg 1021 cccccttcct gcagcagcct gcagccccct gacccgcctgccagctccag tggtgctccc 1081 cacctgagag ccaggaagtc tgagtgggtg tgaHuman Cx 62 (SEQ ID NO: 31)LOCUS      HSA414565   1632 bp   DNA   linear            PRI 30-NOV-2001DEFINITION Homo sapiens CX62 gene for connexin62. 1atgggggact ggaacttatt gggtggcatc ctagaggaag ttcactccca ctcaaccata 61gtggggaaaa tctggctgac catcctcttc atcttccgaa tgctggtact tcgtgtggct 121gctgaggatg tctgggatga tgaacagtca gcatttgcct gcaacacccg gcagccaggt 181tgcaacaata tctgttatga tgatgcattc cctatctctt tgatcaggtt ctgggtttta 241cagatcatct ttgtgtcttc tccttctttg gtctatatgg gccatgcact ttataggctc 301agggcctttg agaaagacag gcagaggaaa aagtcacacc ttagagccca gatggagaat 361ccagatcttg acttggagga gcagcaaaga atagataggg aactgaggag gttagaggag 421cagaagagga tccataaagt ccctctgaaa ggatgtctgc tgcgtactta tgtcttacac 481atcttgacca gatctgtgct ggaagtagga ttcatgatag gccaatatat tctctatggg 541tttcaaatgc acccccttta caaatgcact caacctcctt gccccaatgc ggtggattgc 601tttgtatcca ggcccactga gaagacaatt ttcatgcttt ttatgcacag cattgcagcc 661atttccttgt tactcaatat actggaaata tttcatctag gcatcagaaa aattatgagg 721acactttata agaaatccag cagtgagggc attgaggatg aaacaggccc tccattccat 781ttgaagaaat attctgtggc ccagcagtgt atgatttgct cttcattgcc tgaaagaatc 841tctccacttc aagctaacaa tcaacagcaa gtcattcgag ttaatgtgcc aaagtctaaa 901accatgtggc aaatcccaca gccaaggcaa cttgaagtag acccttccaa tgggaaaaag 961gactggtctg agaaggatca gcatagcgga cagctccatg ttcacagccc gtgtccctgg 1021gctggcagtg ctggaaatca gcacctggga cagcaatcag accattcctc atttggcctg 1081cagaatacaa tgtctcagtc ctggctaggt acaactacgg ctcctagaaa ctgtccatcc 1141tttgcagtag gaacctggga gcagtcccag gacccagaac cctcaggtga gcctctcaca 1201gatcttcata gtcactgcag agacagtgaa ggcagcatga gagagagtgg ggtctggata 1261gacagatctc gcccaggcag tcgcaaggcc agctttctgt ccagattgtt gtctgaaaag 1321cgacatctgc acagtgactc aggaagctct ggttctcgga atagctcctg cttggatttt 1381cctcactggg aaaacagccc ctcacctctg ccttcagtca ctgggcacag aacatcaatg 1441gtaagacagg cagccctacc gatcatggaa ctatcacaag agctgttcca ttctggatgc 1501tttctttttc ctttctttct tcctggggtg tgtatgtatg tttgtgttga cagagaggca 1561gatggagggg gagattattt atggagagat aaaattattc attcgataca ttcagttaaa 1621ttcaattcat aa

Various aspects of the invention will now be described with reference tothe following experimental section which will be understood to beprovided by way of illustration only and not to constitute a limitationon the scope of the invention.

The following Examples are offered by way of illustration and not by wayof limitation.

Example 1 In Vivo Analysis Materials and Methods Laser Treatment

Female Wistar rats (d32-34) were raised under conditions consistent withthe ARVO Resolution on the Use of Animals in Research. Animals wereanaesthetised by administrating a 1:1 mixture of Hypnorm™ (10 mg/ml,Jansen Pharmaceutica, Belgium) and Hypnovel® (5 mg/ml, Roche productsLtd, New Zealand) at a dose of 0.083 ml/100 g body weight in theperitoneum of the animal.

Excimer laser treatment was performed through the intact epitheliumusing a Technolas 217 Z excimer laser (Bausch & Lomb Surgical, USA). Theeye was centered at the middle of the pupil and ablation was performedwith the following parameters: treatment area was of 2.5 mm diameter andof 70 μm depth. This resulted in the removal of a small thickness of theanterior stroma and of the whole epithelium. Excimer laser treatment waspreferentially used to produce reproducible lesions and investigate theeffects of connexin43 AS ODNs on corneal remodeling and engineeringafter trauma.

Following surgery, all animals were placed in individual cages andclosely monitored for any discomfort. Post-surgical in vivo evaluationwas achieved using a slit lamp biomicroscope and/or a slit scanning invivo confocal microscope.

Slit Scanning In Vivo Confocal Microscopy

Prior to, and following corneal laser treatment, each animal wasobserved clinically using a Confoscan 2 (Fortune Technologies America,USA) slit scanning in vivo confocal microscope. The Confoscan 2 is avariant of slit scanning technology with the distinct advantage ofdirect digitization of the images at the time of acquisition. Animalswere anaesthetized and each of them was placed onto a specially designedplatform that was adjusted at the level of the in vivo confocalmicroscope objective lens in front of the acquisition head.

The slit scanning in vivo confocal microscope allows optical dissectionof the living cornea at different levels through the whole cornealthickness. The examination starts from the endothelium and the number ofthe antero-posterior sections depends upon the customized settings. Theslit scanning technology utilizes an objective lens that moves back andforward along the axis perpendicular to the examined area. In brief, thehardware consists of a halogen lamp (100 W/12V), two slits, two tubelenses, a front objective lens, and a highly sensitive digital (CCD)camera. Prior to scanning, a drop of Viscotears (CIBAVision Ophthalmics)is placed on the tip of the objective lens as an immersion substance.During scanning, the eye of the animal is held wide open and orientatedso that the corneal plane is always perpendicular to the optical axis ofthe magnification lens (40×, N.A 0.75). The image acquisition time isapproximately 14 seconds. The gel, not the objective lens contacts theeye at all times. For the rat cornea, up to 250 sequential digitalimages were obtained per examination, and were directly saved to a harddisk drive. Acquisition parameters were adjusted during the preliminaryexperiments and were kept constant for all subsequent experiments. Theywere as follows: the light intensity was decreased to half the intensitygenerally used for human patients, four passes (one pass is consideredas being a full back and forward movement) were used, and a 400 μmworking distance was selected. For the rat cornea, centration isfacilitated by clear visualization of the pupil, which provides verygood topographical repeatability.

In Vivo Confocal Images

All images acquired with the slit scanning in vivo confocal microscopewere stored onto the hard disc drive and subsequently analyzed by NAVISproprietary software (Confoscan 2, Nidek Co Ltd).

Stromal dynamics were evaluated following stereological principles. Cellcounts were recorded at the anterior and posterior stromal positions.The main stereological component was provided by the in vivo confocalmicroscope itself as it functions as an optical dissector (a probe thatsamples with equal probability particles in space). Indeed, the in vivoconfocal microscope provides thin optical slices of specified volume,with each being a dissected tissue sample. As a result, counting stromalcells consists of choosing a pair of frames (consecutive picturesrecorded by the in vivo confocal microscope), one frame having particles(stromal cells) in focus, and the co-frame showing a defocused butrecognizable image of the same particles (optical shadows). The numberof cells (n) is recorded from the clearest frame in a defined area A(μm²). The distance d (μm) between the two frames is also recorded. Thenumber of cells per unit volume (V) therefore equals to: V=Number ofcells (n)/d (μm)×A (μm²).

Ex Vivo Confocal Images

Appropriate corneal sections were immunohistochemically stained withdifferent markers for different purposes. Staining with the nuclearstain Hoechst 33 258 was used to estimate the number of epithelial andstromal cells at the central or the peripheral cornea. For this purposeusing AnalySIS® 3.2 software (Soft Imaging System, USA), the area ofinterest was first freehand drawn onto the TIFF file image of theappropriate region of the cornea and the value of the area wasautomatically given by the software. Using the manual count option,cells were then counted within that area and expressed per unit area.

Antisense Compound Application

30% Pluronic acid gel (BASF Corp) in phosphate buffered saline(molecular grade water) was used to deliver unmodified α1 connexin(connexin43) specific antisense ODNs to the subconjunctiva ofanaesthetized rats following photorefractive keratectomy. In a pre-trialusing an FITC tag, this formulation was shown to remain in the anteriorchamber of the eye for up to 24 hours (not shown).

The antisense molecule used in these experiments was DB1 ((GTA ATT GCGGCA GGA GGA ATT GTT TCT GTC) (SEQ ID NO: 65). Addition of an FITC tag toDB1 ODN, viewed using confocal laser scanning microscopy, demonstratedintracellular penetration of the probe.

The ODN was applied at a 2 μM final concentration.

Monitoring Tissue Engineering or Remodeling Effects

After antisense application, the corneas were examined using a slitscanning in vivo confocal microscope at 2 h, 12 h, 24 h, 48 h, 72 hr, 1week and 2 weeks post laser surgery. Control rats received laser surgeryonly.

Table 1 summarizes the number of corneas investigated at each timepoint.

TABLE 1 Number of control (C) and AS (ODN) treated corneas used for thein vivo follow-up using slit scanning in vivo confocal microscopy. 1week 2 weeks Within 2 hr 12 hr post- 24 hr post- 48 hr post- 72 hr post-post- post- surgery surgery surgery surgery surgery surgery surgeryNumber of 18 C 10 C 18 C 10 C 6 C 4 C 5 C eyes (n) 18 ODN 10 ODN 18 ODN6 ODN 6 ODN 4 ODN 5 ODN ODN = AS ODN treated eyes (single administrationafter laser surgery)

Each cell layer of the cornea was analyzed and the cell type, number andappearance compared between the control and ODN treated groups.

Re-Epithelialization:

Treatment with anti-connexin43 ODNs promoted epithelial recovery. In 90%of AS ODN treated corneas, sliding epithelial cells were observed within12 hours after PRK laser surgery, compared to none in controls (FIG.1B). At this stage, only static endothelial cells were present in 30%control corneas (FIG. 1A) By 24 hours epithelial cells were seen in allcontrols and antisense treated corneas but 72% of treated versus 61% ofcontrols showed actively sliding cells. This indicates thatre-epithelialization is proceeding faster in the connexin43 specific ASODN treated corneas than in controls.

Stromal Cell Densities:

Using a paired samples t-test with repeat measures to compare celldensities in the anterior and posterior stroma within each group as afunction of time and a Mann Whitney non parametric statistical test tocompare stromal cell counts between control and ODN treated corneas atthe selected time points, the only statistically significant resultswere found at 24 hr post-laser surgery (Table 2). At this time point, inthe control and ODN treated groups stromal cell density in the anteriorstroma has increased considerably compared to the pre-surgery values (pvalue <0.05). In the posterior stroma of control corneas, stromal celldensity has also increased compared to the pre-surgery value (p value<0.05) whilst in the posterior stroma of ODN treated corneas, stromalcell density is not statistically significantly different from thepre-surgery value (p value >0.05). When comparing stromal cell densitybetween the two groups at the anterior and posterior stroma, the ODNtreated corneas always showed lower stromal cell densities than thecontrol corneas (p-value <0.05). This supports the idea that a smallernumber of cells are involved in stromal re-modeling or engineering inthe ODN treated corneas compared to the control corneas. This is thefirst report showing that application of anti-connexin43 ODNs reduceshypercellularity at the site of surgery. Ex vivo histochemical analysis(Example II) shows that this hypercellularity is associated withmyofibroblasts which induce unwanted stromal matrix remodeling andscarring.

TABLE 2 Stromal cell counts in control and AS ODN treated corneas priorto and 24 hr following photorefractive keratectomy. Cell densities aregiven as means followed by standard deviations. Anterior stromal cellPosterior stromal cell count count Treatment Time points (#cells/mm³)(#cells/mm³) Control Pre-surgery 36469 ± 11122 33909 ± 8753 (n = 17) (n= 17) ODN Pre-surgery 36769 ± 10932 34382 ± 8667 (n = 14) (n = 17)Control 24 hr post- 144643 ± 60989 46901 ± 26964 (n = 17) surgery (n =17) ODN 24 hr post- 93468 ± 53548 33510 ± 11350 (n = 14) surgery (n =17)

Example II Ex Vivo Analysis Materials and Methods Histology: TissueCollection and Fixation

Appropriate numbers of animals (Wistar rats) were terminated at selectedtime points following photorefractive keratectomy and DB1anti-connexin43 ODNs were administered to anaesthetized rats asdescribed in experiment 1 above and corneal sections were prepared forhistological analysis. Control rats had received laser surgery only.Whole eyes and control tissues were rinsed in Oxoid PBS prior toembedding in Tissue-Tek® OCT (Sakura Finetek, USA) and freezing inliquid nitrogen. When necessary (for the use of some antibodies), frozentissues were later fixed in cold (−20° C.) acetone for 5 min after beingcryocut.

Tissue Cutting

The procedure for cryosectioning was as follows: frozen blocks ofunfixed tissue were removed from −80° C. storage and placed in the LeicaCM 3050S cryostat for about 20 min to equilibrate to the sametemperature as the cryostat (i.e. −20° C.). When equilibration of thetissue was achieved, the specimen was mounted onto a specimen disc withTissue Tek® OCT. Sections of 12 μm (for H/E staining) or 25 μm thick(for immunolabeling) were cut and placed on Superfrost®Plus slides(Menzel-Gleser, Germany). Immediately following cryocutting, tissueblocks were placed back to −80° C. storage and slides supportingcryosections were either used immediately or stored at −80° C.Sectioning occurred parallel to the optical axis of the eye.

Haematoxylin/Eosin (H/E) Staining and Nuclear Staining

Slides were placed in glass racks to facilitate immersion in a series ofdifferent staining reagents. Racks were agitated when placing them intoreagents to break surface tension and to drain them between eachsolution change. Prior to Gill's II Haematoxylin/Eosin staining, slidesthat were stored at −80° C. were first warmed up to room temperature for1-2 min, then either fixed in cold acetone first and/or immediatelyhydrated with a quick dip in tap water. Slides were stained in Gill's IIHaematoxylin for 2 min, after which excess stain was rinsed off in tapwater. Stain differentiation was achieved by dipping in Scott's tapwater substitute (STWS) for 4 sec. A rinse in running tap water for 1min was then performed before staining in 1% eosin for 30 consecutivedips. Finally, sections were quickly rinsed in tap water, dehydratedthrough 95%, 100% EtOH, cleared in xylene, and mounted with DPX mountingmedium (Sigma). For nuclear counter staining (in parallel with H/E orimmunohistochemical analysis) Hoechst 33 258 (Sigma) was used.Measurement of cornea thickness was carried out on H/E stained sections.

Immunohistochemistry

Sections were immunolabeled for connexin43 using a site-specificmonoclonal antibody, for myofibroblasts using an antibody recognizingalpha smooth muscle actin, for basal lamina deposition with ananti-laminin-1 antibody. In addition anti-vimentin antibodies were usedto differentiate stromal keratocytes from myofibroblasts and a Ki-67antibody was used to show cell proliferation.

Ex Vivo Histological Analysis

Results showed that lesions made by excimer photoablation had closed by24 hr post-surgery (FIG. 2). The typical invasion of the stroma bymononucleated/multinucleated and/or round, ovoid cells at the peripheryand at the center of the cornea was observed in both groups, mostpronounced at 24 hr post-surgery, but with the antisense ODN treatedgroup showing a significantly smaller number of these cells than thecontrol group (FIG. 2A,B,C). This parallels the findings from the invivo confocal photomicrographs shown in Example 1. The epitheliumthickness was variable in control corneas as seen in FIG. 2 at the siteof laser induced lesion (Figure A, B), and in the stoma beneath theablated area (Figure A,B) and in the peripheral stroma (Figure C) therewas an extensive invasion by round cells (hypercellularity) in controlcorneas. Also observed was a pronounced stromal edema in Figure B andFigure C. In the antisense ODN treated corneas the epithelium was ofeven thickness (Figure D,E) and in the central region (Figure D) and inthe peripheral stroma (Figure E) there was little sign of stromal edema.Moreover, in the stroma there were few round cells present. Scales barsin FIG. 2 represent 20 microns.

Changes in stromal thickness following treatment with connexin43 ODNsafter laser treatment are shown in Table 3, which compares changes instromal thickness between control and ODN treated corneas. Stromalthicknesses were measured from appropriate histological stainedsections. Statistical analysis of the data obtained for the ODN treatedgroup using a paired samples t-test showed that at all three time pointsinvestigated (24 hr, 48 hr and 72 hr post-surgery) central stromalthickness is statistically significantly thinner than pre-surgery value(p values <0.05) and peripheral stromal thickness is not significantlydifferent from pre-surgery values. In contrast, control corneas showsignificant stromal swelling (edema) (FIG. 2 A, B, C) in both centraland peripheral cornea (where the stroma doubles in thickness compared topre-surgery values).

TABLE 3 Changes in stromal thickness following excimer laser surgery incontrol and AS treated corneas. mean central mean peripheral stromalthickness stromal thickness Treatment Time points (μm) (μm) Normal (nosurgery) Pre-surgery  250 (n = 6)*  110 (n = 10) Control 24 hr post- 318(n = 6) 290 (n = 6) ODN treated surgery 190 (n = 5) 132 (n = 5) Control48 hr post- 307 (n = 6) 206 (n = 5) ODN treated surgery 158 (n = 5) 105(n = 5) Control 72 hr post- 292 (n = 6) 201 (n = 6) ODN treated surgery142 (n = 5)  99 (n = 5)

Cornea which is not subjected to surgery had a central stromal thicknessof 250 μm, but excimer laser surgery was used to remove 70 μm of cornealtissue (including the epithelium and part of the stroma). The normalcorneal epithelium is 50 μm thick (on average) and therefore 20 μm ofstromal tissue was removed by laser surgery. Therefore, to statisticallycompare the central stromal thickness at 24 hr, 48 hr and 72 hrpost-wounding to the pre-surgery central stromal thickness, an adjustedthickness loss and a central pre-surgery stromal thickness of 250−20=230μm was used.

Reduction in Connexin43 Expression is Associated with Reduced StromalInvasion and Reduced Epithelial Hyperplasia

Microscopial observations showed a reduced level of connexin43 presentin ODN treated corneas compared to control corneas. FIG. 3 showscombined micrograph images. Top row shows control corneas, the bottomrow shows antisense ODN treated corneas. The typical invasion of thestroma by round cells was seen in both groups within 24 hours at thelimbal, peripheral and central areas. However, a smaller density ofround cells was exhibited in ODN treated corneas. At the limbus in bothgroups anti-connexin43 was evenly distributed throughout the stroma(3A,D) but the treated groups had less label in the periphery (3E)compared to controls (3B). By this stage connexin43 levels had returnedto normal in the epithelium of both groups but control groups showed ascar like stroma (3C) or hyperplasia (see FIG. 4 below) whereas inantisense treated corneas a normal epithelium with normal levels ofconnexin43 was seen (3F). Scale bars A, D, E, F represent 10 microns; Band C represent 20 microns. In these figures connexin43 appears as whitepunctate labeling with cell nuclei appearing grey. The results shown inFIG. 3 suggest that connexin43 protein levels are reduced followingtreatment with anti-connexin43 ODNs and results in a smaller degree ofcell recruitment in the stroma. In addition, only 7% of ODN treatedcorneas (0% at 24 hr post-surgery, 0% at 48 hr post-surgery, 20% at 72hr post-surgery) show signs of epithelial hyperplasia compared to 31%control corneas (25% at 24 hr post-surgery, 67% at 48 hr post-surgery,0% at 72 hr post-surgery). This was assessed on H/E stained and Ki-67labeled sections.

Myofibroblast Labeling

Labeling with vimentin antibodies indicated that the increased cellnumbers in the stroma of control corneas compared with AS ODN treatedcorneas were not of undifferentiated keratocyte origin and labeling wastherefore carried out with alpha-smooth muscle actin antibodies. Thislabeling showed that control corneas had a higher number ofmyofibroblasts beneath the site of surgery, but also in the surroundingperipheral stroma. This increase in myofibroblast numbers and areaaffected was evident at 24 hours and persisted over 48 and 72 hoursthrough to at least one week after surgery (Table 4). FIG. 4 showsmyofibroblast labeling (anti-alpha smooth muscle actin) at 1 weekpost-laser surgery. FIGS. 4 A, B, and C are controls; and FIGS. 4 D, E,and F are antisense treated corneas. By one week post-wounding, in thecontrol corneas, low to moderate numbers of myofibroblasts are presentin the anterior half of the peripheral stroma (4A), moderate to denselevels are present in the mid-peripheral stromal regions (4 B), andmoderate levels are seen in the anterior half of the stroma in centralregions (4C). In contrast, in the treated corneas, very low numbers ofmyofibroblasts are present in peripheral (4D) or mid peripheral (4E)stroma and moderate to low numbers in central stroma (4F). In some casesin the central stroma, myofibroblasts are concentrated in the area justunder the epithelium (not shown). Thus, the increased cell numbers seenin Example 1 (hyerpcellularity) and FIG. 2 above appears to be due tomyofibroblast differentiation and invasion. Myofibroblasts are known tobe responsible for scar tissue deposition in the stoma, with reducedcrystalline deposition and increased secretion of wound collagen III(Ahmadi A. J. and Jakobiec F. A.; 2002; Int Ophthalmol Clin. Summer;42(3):13-22).

TABLE 4 Summary of alpha smooth muscle actin labeling for myofibroblastin control and antisense ODN treated corneas. Time Locations Controlcorneas AS treated corneas 24 hr post-surgery Periphery 80% D in wholest 100% M in anterior 20% M in whole st 1/3 st Mid-periphery 100% D inwhole st 100% M in anterior 1/3 st Centre 100% L in anterior ¾ st 80% Lin anterior ⅓ st 20% L below epi 48 hr post-surgery Periphery 83% M inwhole st 40% M in half 17% L in whole anterior st 60% L in half anteriorst Mid-periphery 50% D in whole st 100% D in half 50% M in whole stanterior st Centre 17% L under epi 20% absent (hyperplasia) 80% M inanterior ¾ st 50% D in whole st 33% M in whole st 72 hr post-surgeryPeriphery 33% L in anterior half st 40% L in anterior 67% M in anteriorhalf st half st 60% M in anterior half st Mid-periphery 33% L in wholest 20% L in anterior 67% Din whole st half st 80% M in anterior half stCentre 17% absent 20% absent 50% D in whole st 40% L in anterior half st33% M in whole st 40% M in anterior ¾ st 1 week post-surgery Periphery60% M in half anterior st 100% L in anterior 40% L in half anterior st1/3 st Mid-periphery 60% D in whole st 100% L in anterior 40% M in wholest half st Centre 60% M in anterior half 60% M in anterior st half st40% L in anterior half st 20% L in anterior half st 20% M under epiNumbers of myofibroblasts are quantified as dense (D), moderate (M), low(L) or absent. Percentages refer to proportions of animals affected atthe specified levels. st = stroma, epi = epithelium. Significantdifferences between control and antisense treated corneas arehighlighted in bold.

Basal Lamina Deposition

Following photorefractive keratectomy the basal lamina reforms alongwith the regrowing epithelium. Labeling with antibodies to laminin-1shows that the reforming basal lamina is discontinuous and with anirregular epithelial-stromal attachment (FIG. 5). At 24 hours controlshad little and/or uneven laminin deposition at the edge of the ablatedarea (FIG. 5A) and more centrally (FIG. 5B) whereas antisense treatedcorneas showed a more regular deposition of laminin at both of theseregions (FIG. 5C, FIG. 5D). At 48 hours controls still do not have acontinuous laminin deposition (FIG. 5E—edge of the ablated area; FIG.5F—central) and it was very uneven (FIG. 5E). In contrast antisense ODNtreated corneas had a continuous and relatively even basal lamina at thewound edge (FIG. 5G) and centrally (FIG. 5H). All scale bars in FIG. 5represent 20 microns. Connexin43 antisense treated corneas formed adenser, more continuous basal lamina within 24 hours with lessirregularity.

The laminin irregularity was quantified as shown in FIG. 6. The blacksolid line in FIG. 6 represents laminin-1 deposition. For each regionthe variance was measured as the difference between the top of a hilland the bottom of a valley (FIG. 6 A, B, C, D). Control corneas had amean variance of 6.98 microns compared with 4.74 microns in antisenseODN treated corneas. The difference between the two groups wasstatistically significant (p<0.0001).

Example III Ex Vivo Tissue Engineering

Corneas were placed into an ex vivo organ culture model and specificconnexin modulated using antisense ODNs. Two connexins were targeted inthese experiments, connexin43 and connexin31.1. Connexin43downregulation is used to demonstrate that connexins can be regulated invitro, and connexin31.1 was targeted because this connexin is expressedin the outer epithelial layers of the cornea in cells about to be shedfrom the cornea. The aim was to engineer a thickening of epithelialtissue by reducing connexin31.1 expression.

Materials and Methods

30-34 day old Wistar rats were euthanized with Nembutal or carbondioxide and whole rat eyes dissected. The ocular surface was dissected,disinfected with 0.1 mg/ml penicillin-streptomycin for 5 minutes andrinsed in sterile PBS. The whole eye was then transferred onto a sterileholder in a 60 mm culture dish with the cornea facing up. The eyes weremounted with the corneal epithelium exposed at the air-medium interfaceand cultured at 34° C. in a humidified 5% CO₂ incubator in serum freemedium (Opti-MEM, Invitrogen) for up to 48 hours. 100 μl of medium wasadded drop wise to the surface every eight to twelve hours to moistenthe epithelium. Medium levels were maintained to the level of limbalconjunctiva.

Antisense oligomers were mixed with 30% (w/w) Pluronic F127 gel (Sigma)on ice to a final 2 μM concentration and 10 μl applied onto the corneas.Each treatment had a sample size of 3 to 4 corneas per experiment.Preliminary experiments showed that double treatments of our positivecontrol, DB1, for 8 hours had little effect on connexin43 proteinexpression in our corneal culture. Corneas were therefore cultured for24 hours and connexin specific oligomers applied every 8 hours.

Immunohistochemical labeling was carried out as in Experiment 2 aboveusing antibodies to connexins43, 26 (control) and 31.1. Tissue was alsostained with H/E as above. Nuclei were counterstained with 0.2 μMpropidium iodide. Images were collected on a Leica TCS 4D or TCS SP2confocal laser scanning microscope with voltage and offset settingsmaintained within experimental groups to allow quantification ofconnexin levels. For quantification four optical slices through 3microns were processed into a single extended focus optical image usingthe center of mass topographical projection option on the TCS 4D.Connexin label was quantified using NIH Image (Scion Corp. USA) afterthresholding at 90-100 pixel intensity on the 256 grey scale image.

In corneas that have not undergone surgery, in vitro connexin turnoverrates were relatively low compared to tissue remodeling processes in theexcimer laser ablated corneas described in Examples 1 and 2 hereinabove. Nonetheless, after three treatments with antisense ODNs connexinlevels were reduced by over 50% compared with controls (FIG. 7 A, Bshows connexin43 reduction in AS ODN treated corneas compared withcontrols). Connexin26 levels remained constant when the connexin43specific antisense ODNs were applied (indicating that the reduction inconnexin levels was specific, not a side effect of the treatment. Inthese images connexin43 appears as heavier spots in the basal two layersof the epithelium, connexin26 as finer punctate labeling predominantlyin layers 2-6. Connexin31.1 antisense ODNs reduced levels ofconnexin31.1 but preliminary results also showed that the epithelialthickness (number of layers) increased within 24 hours (FIG. 7 C, D).This increase in thickness was seen using H/E staining (FIG. 7D) and inthe immunohistochemically (FIG. 7C) labeled sections.

The results described in this work form a basis for the use of connexinspecific antisense ODNs in tissue-engineering, including specificallyafter excimer laser surgery of the cornea, or for in vitro organ culturefor tissue engineering and transplantation. The experimental resultsprovided herein demonstrate that a single treatment with connexin43specific antisense ODNs following excimer laser photorefractivekeratectomy has many beneficial uses, some of which are describedhereinbelow.

Administration of connexin specific antisense ODNs promote epithelialcell movement. At 12 hr post-surgery 90% antisense treated corneas butno control corneas show the presence of sliding epithelial cells at thesite of a laser induced lesion. Epithelial cells were present in 30% ofcontrol corneas but were static/non-sliding. Regulation of directcell-cell communication by connexins can therefore be used to engineerchanges in epithelial cell patterning.

Administration of connexin specific antisense ODNs promote controlshypercellularity associated with myofibroblast differentiation at thesite of a laser induced lesion in the 24 hr to 48 hr post-surgeryperiod. During this period, more control corneas (63%) than antisenseODN treated corneas (39%) show intense hypercellularity in the wholestroma. Regulation of direct cell-cell communication can therefore beused to modulate cell differentiation leading to modification ofextracellular matrix.

Administration of connexin specific antisense ODNs controls stromalremodeling reducing haze at the site of a laser induced lesion in the 24hr to 72 hr post-surgery period. In this period, more control corneas(64%) than antisense treated corneas (39%) show intense haze in thewhole stroma.

Administration of connexin specific antisense ODNs inhibits stromaledema during the early stages of re-modelling. Regulation of directcell-cell communication therefore improves outcomes from laser surgery.

Administration of connexin specific antisense ODNs reduces cellproliferation in the early stages of re-modelling. Regulation of directcell-cell communication can therefore be used to regulate cellproliferation during tissue remodeling.

Administration of connexin specific antisense ODNs reduces epithelialhyperplasia by 78% (assessed from 24 hr to 72 hr post-surgery) enablingengineering of an even epithelium.

Administration of connexin specific antisense ODNs reduces myofibroblastactivation up to 1 week post-surgery (and earlier loss of keratocytes).Regulation of direct cell-cell communication enables more precisecontrol of tissue damage during surgical remodeling, providing improvedpredictability of outcome and fewer visual defects.

Administration of connexin specific antisense ODNs results in a moreregular and denser epithelial-stromal adhesion matrix during tissuere-modelling. Regulation of direct cell-cell communication can thereforebe used to engineer tissue basal laminae.

In addition, the ex vivo corneal culture model used herein indicatesthat regulation of direct cell-cell communication can be used toengineer tissue in vitro, for example increasing epithelial thicknessusing connexin31.1 antisense ODNs. This treatment also has implicationsin vivo, for example in the engineering a thicker cornea for the reliefof corneal diseases such as keratoconus (a thinning of the epithelium).

The results show that active molecules which interfere with cell-cellcommunication can be used in tissue engineering and remodeling.Specifically, it is shown that antisense deoxynucleotides targeted atconnexin proteins can be used in corneal re-modeling especiallyfollowing corrective laser surgery, as well as for in vivo and in vitrotissue engineering.

The antisense compounds and methods described herein therefore havesignificant potential for improving the outcome of surgicalinterventions and ameliorating disease processes in the eye, and fortissue engineering.

Example IV Ex Vivo Culture Model

Application of antisense oligodeoxynucleotides specific to the gapjunction protein Connexin43 following brain or spinal cord injury inadult animal models blocks lesion spread, and reduces the inflammatoryresponse and subsequent scar formation. We have taken our antisenseapproach even further and developed an ex vivo culture model for spinalcord segments and intact cords in order to elaborate repair strategiesfor established lesions.

Spinal cords are excised from P7-P14 rat pups and divided into caudal,thoracic and rostral segments. Antisense oligodeoxynucleotides wereapplied in a Pluronic gel to the cut ends of the spinal cord segmentsduring placement in culture, this results in a reduction of Connexin43protein levels for 24-48 hours, significantly improving viability of thetissue. The most immediate and notable observation is that swelling doesnot occur (FIG. 8 A-B, showing cord segments 24 hours after placing intoculture). This treatment blocks the spread from the spinal cord cutsends. Increased neuron survival in the grey matter of treated samplesare clearly evident in the toluidine blue-stained resin sections (FIG.9A). In sharp contrast, edema and vacuolation of neurons is seenthroughout control tissue (untreated, gel only or gel with randomoligodeoxynucleotides) in FIG. 9B.

Subsequent labeling and immunohistochemical studies up to day 20 showthat neurons in the treated cord segments (Neuronal-N labeling) survivefor this period whereas few remain viable after as little as 3 days inthe control segments. Isolectin-B4 labeling shows extensive activated(macrophagic phenotype) microglial invasion of control segments withinfive days in culture. In treated samples, activated microglial cells arerestricted to the outer edges (where the white matter axon tracts werepreviously, and at the very cut ends). Notably, MAP-2 labeling, a markerfor neuronal processes, indicates significant potential for regrowth intreated cord segments compared to control segments, which show no MAP-2labeling at all (FIG. 10A-B).

Example V Grafting of Peripheral Nerves Across Spinal Cord Lesions

For peripheral nerve grafting, we will retreat the tissue withConnexin43 specific antisense oligodeoxynucleotides at the time ofgrafting to prevent lesion spread from the graft site and microglialactivation which leads to isolation of the graft from the host tissueneural population, restricting neuronal repair.

Peripheral Nerve Grafts:

Spinal cord segments are placed onto culture inserts (MilliporeMillicell) in 35 mm dishes and the level of the culture medium raiseduntil a miniscus forms over the segments. Connexin43 specific antisenseoligodeoxynucleotides (30mers, 1 μM concentration) in a 30% Pluronic gelare placed immediately over the cord tissue. The gel sets as it warms tophysiological temperatures and provides sustained release of theantisense oligomers. This treatment will reduce connexin43 proteinlevels for between 24 and 48 hours, with maximum reduction at 6-8 hourspost-treatment. Such cord segments, stabilized in culture, and thenre-exposed to incision trauma, shows the same symptoms as surgicalintervention in vivo, including lesions expansion and tissue swellinginto the cut area. This effect can be prevented by treatment withConnexin43 specific antisense oligomers at the time of the incision; andaccordingly, the cut edges remain sharply defined with no obvious signsof edema or tissue swelling.

The segments are placed end to end but separated by a gap of 1-5 mm.After a one to three day stabilization period in culture, a graft of asciatic nerve from a P7-P14 rat pup is placed across the gap. Previousstudies have indicated that both sciatic nerve (or its saphenous branch)(Yick, L. W. et al., 1999, Exp Neurol. 159: 131-138; Aguayo, A. J. etal., 1981, J. Exp. Biol. 95: 231-240) or intercostal nerve (Cheng, H.,et al., 1996, Science, 273: 510-513) grafting has considerable potentialto induce axon elongation and the survival of neurons.

Immediately after grafting, re-treatment will commence with theConnexin43 antisense oligomers accompanied by neuronal behaviorassessment over the subsequent days. Since the culture period aftergrafting are relatively short (up to 15 days) compared with in vivostudies (15 days to 7 months after surgery) a variety of markers toassess repair response as detailed below (Measuring outcomes) are used.Experiments are conducted with and without the addition of exogenousgrowth factors (such as acidic FGF or NGF) which might play a role ininducing neuronal proliferation and/or migration.

Example VI Insertion of Schwann-Cell-Seeded Implants Between SegmentsPlaced End to End

For implants between treated segments, Schwann cells have been selectedas they have been shown to be strong promotors of axonal regeneration(Xu, X. M. et al., 1999, J. Neurosci. 11: 1723-1740; Keirstead, H. S. etal., 1999, Exp. Neurol. 159: 225-236).

Implanted cells can provide a permissive environment for central nervoussystem axon regeneration and have proven to be effective for inducingregrowth of axons. Schwann-cell-seeded mini-channel implants or matrigelplaced between cord segments placed end to end are to be used for thisapplication. The principle here is that for spinal repair interventions,one would ultimately wish to excise scar tissue and fill the space withimplant material. The methods used are described by Morrissey et al. andXu et al. (Morrissey, T. K. et al., 1991, J. Neurosci. 11: 2433-2442;Xu, X. M. et al., 1995, J. Comp. Neurol. 351: 145-160). Essentially,sciatic nerves are obtained from adult rats, the epineurium andconnective tissue are then removed and 1 mm long explants are placedinto culture with Dulbecco's Modified Eagle's Medium (DMEM-Gibco, USA).Outgrowth of migratory cells are predominantly fibroblasts and theexplants are moved to a new dish as these reach confluency. This isrepeated over three to five passages until the cells that emerge areprimarily Schwann cells. These are dissociated and grown up for seedinginto copolymer or matrigel guidance channels (Schmidt, C. E. and BaierLeach, J., 2003, Ann. Rev. Biomed. Eng. 5: 293-347). Once implantmaterial is prepared, cultured segments will have their ends recut tomimic scar excision, and placed end to end with implant material wedgedbetween. Immediately after grafting, samples are re-treated with theConnexin43 antisense oligomers and the neuron behavior is monitored oversubsequent days.

Measuring Outcomes

Time course experiments are carried out for both peripheral grafts andimplants to establish whether there is immediate, late or continuousresponse to the graft tissues. Several markers are used to assessneuronal response and repair potential. These include: Neuronal(antibodies to Neuronal-N), neurofilament (antibodies to MAP-2 andSMI-31) and cytoplasmic markers (CMFDA) and membrane dyes (Di-I or Axongrease-Molecular Probes, Oregon, USA). Increased neural sprouting,increased axon migration distance (bridge length to distance migratedratio) and increased numbers of axons growing toward or across the graftare specifically monitored. Cell specific markers (GFAP for astrocytes,Isolectin-B4 for microglial cells, and S-100 for Schwann cells). Glialcell distribution and density, and levels of myelination are assessed.Anti-CGRP (a peripheral nerve marker) are used to distinguish betweenaxons of peripheral nerve origin as opposed to those regenerating fromthe cord segments. GAP-43 (growth associated protein) antibodies areused to identify neuronal growth cones. Toluidine blue stained semithinsections and electron microscopy of graft cross sections are used formorphological analysis.

Secondary antibodies are conjugated with Alexa dye. For double or triplelabeling, we use Zenon probes (Molecular Probes, Oregon, USA) whereappropriate. All antibody and dye labels are analysed using TheUniversity of Auckland's Biomedical Imaging Research Unit Leica TCS 4Dand SP2 confocal laser scanning microscopes. Electron microscopy isperformed on a Hitachi H-7000 electron microscope. Image analysisprogrammes (AnalySIS or NIH Image J) are used to quantify differencesbetween control and treated grafts.

Example 7 Antisense Oligodeoxynucleotide Design Materials

Materials used herein include art-recognized antibodies and plasmids;such as, for example, plasmids for rat connexin 43 (T7291),) andconnexin 26; plasmids for mouse connexin 43 and connexin 26 (Invivogen,USA), mouse anti rat connexin 43 and rabbit anti rat connexin 26 fromZymed (51-2800); and goat anti mouse Alexa 488 and goat anti rabbitAlexa 568 secondary antibodies from Molecular Probes, Eugene Oreg.Nuclei were stained using Hoechst 33258 dye (Sigma). All deoxyribozymesand oligodeoxynucleotides were purchased from Sigma Genosys, Australia,as desalted oligomers. TaqMan labeled oligomers were purchased fromApplied Biosystems, USA. All oligodeoxynucleotides were purchased asunmodified phosphodiester oligodeoxynucleotides.

Deoxyribozyme Design

The deoxyribozyme design and testing was similar to that described inprevious studies (Santoro, S. W. and Joyce, G. F. Proc. Natl Acad. Sci.USA, (1997), 94, 4262-4266 and Cairns, M. J. et al., (1999) Nat. Biotech17, 480-486). In brief, all AU and GU sites in the mRNA sequence of thetarget connexin were selected with 8 or 9 nucleotides on each side ofthe A or G. The deoxyribozymes are the complement of this sense codingsequence with the “A” or “G” replaced with the “10-23” catalytic core“ggctagctacaacga”. Control deoxyribozymes had a defective catalytic coreof “ggctaActacaacga” with a single point mutation (g→A) Santoro, S. W.and Joyce, G. F. Biochem 37, 13330-13342). We also designed GC and ACspecific deoxyribozymes to cover gaps left by AU and GU deoxyribozymesnot meeting the three requirements below. Each deoxyribozyme was namedaccording to the position of “A” or “G” nucleotides from the start ATGcodon. Those deoxyribozymes selected for in vitro assay had to fulfillthree requirements:

1. Thermo stability: the chosen deoxyribozymes should not form stablesecondary structures, either hairpin looping or homodimers. Anydeoxyribozyme with a hairpin or homodimer melting temperature greaterthan 37° C. was discarded as presumptively unable to bind to the targetsequence at physiological temperatures.

2. Affinity: The total ΔG values of both binding arms should not begreater than −30 Kcal. Each individual binding arm is between −10 to −15kcal. This is a compromise between the specificity of binding/misspriming (due to higher CG content) and an effective binding/turnoverrate requirement for deoxyribozymes. The binding arm length either sideof the cleavage site is adjusted to find the ideal ΔG value and step (1)repeated to check.

3. Specificity: All target binding sequences were BLASTn searched withGene Bank to check for specificity (http://www.ncbi.nlm.nih.gov/BLAST/).Deoxyribozymes with homology to other connexin genes or other knownrodent genes were discarded.

In Vitro Testing of Deoxyribozymes

The mouse connexin43 and connexin26 cDNAs were excised from the pORFvector (Invivogen) with NcoI and NheI and subcloned into pGEM-T(Promega) prior to in vitro transcription. Both the full-length 2.4 kbrat connexin43 cDNA and the full coding 1.4 kb rat connexin43 cDNAincluding 200 nucleotides of 5′-untranslated regions were used for invitro transcription. Full length mRNA was transcribed from linearizedplasmid DNA using a Promega Riboprobe Kit. The resulting mRNA waspurified with a PCR spin column (Qiagen). Concentration was determinedby spectrophotometer reading of OD at 260 nm. Deoxyribozymes (40 μMfinal concentration) and mRNA (0.01 to 0.05 μg/μl total mRNA) were thenseparately pre-equilibrated with a 2× cleavage buffer (100 mM Tris 7.5;20 mM MgCl₂; 300 mM NaCl; 0.02% SDS) for 5-10 minutes at 37° C. mRNA anddeoxyribozyme mix were then incubated for one hour at 37° C., followingwhich, 10× Bluejuice (Invitrogen) was added to stop the cleavagereaction and the mixture kept on ice. The reaction mixture was thenloaded onto a pre-run 4% polyacrylamide gel (19:1 acryl:bis ratio,BioRad) in 1×TBE buffer and 7M Urea and run for up to 2 hours. Gels werestained with a 1:10 000 dilution of SYBR green II (Mol Probes, USA) inTBE buffer and imaged using a BioRad Chemi Doc system.

Design of Antisense Oligomers

Antisense sequences were chosen based on the twenty-nucleotide sequencesof the deoxyribozyme binding arms that were successful in cleaving themRNA in vitro. Selected sequences were chosen for use in the design of30-mer oligos (Brysch, W. (1999). Antisense Technology in the VentralNervous System, ed. H. A. Robertson; Oxford University Press 21-41) and(Walton S., et al., (2002) Biophysical Journal 82, 366-377). In brief,sequence related side effects such as partial sequence homology of 8-10CG base pairings to unrelated genes, GGGG and CpG motifs were avoided.Antisense sequences with the 3′-end ending with a Thymidine or more thanthree C or Gs in the last five nucleotides are also avoided if possibleto prevent miss priming. Oligomers that form stable secondary structuressuch as homodimers, palindrome motifs or secondary hairpin structureswill impede oligomers binding to the target mRNA. Control oligomers,including sense, scrambled, reverse and mismatch oligomers were alsodesigned to assess possible chemistry related side effects due to crosshybridization, non specific protein binding, and toxicity.

Corneal Organ Culture and Treatment with Antisense Oligonucleotides

30-34 day old Wistar rats were euthanized with carbon dioxide and wholerat eyes dissected. The ocular surface was dissected, disinfected with0.1 mg/ml penicillin-streptomycin for 5 minutes and rinsed in sterilePBS. The whole eye was then transferred onto a sterile holder in a 60 mmculture dish with the cornea facing up. The eyes were mounted with thecorneal epithelium exposed at the air-medium interface and cultured at34° C. in a humidified 5% CO₂ incubator in serum free medium (Opti-MEM,Invitrogen) for up to 48 hours. 100 μl of medium was added drop wise tothe surface every eight to twelve hours to moisten the epithelium.Medium levels were maintained to the level of the limbal conjunctiva.

Antisense oligomers were mixed with 30% (w/w) Pluronic F127 gel (Sigma)on ice to a final 2 μM concentration and 10 μl applied onto the corneasas previously described. (See Becker, D. L., et al.; (1999b) Dev. Genet.24:33-42; Green, C. R., et al.; (2001), Methods Mol Biol 154, 175-185).Each treatment had a sample size of 3 to 4 corneas per experiment.Preliminary experiments showed that double treatments of our positivecontrol, DB1, for 8 hours had little effect on connexin43 proteinexpression in our corneal culture. Corneas were therefore cultured for24 hours and connexin43 specific oligomers applied every 8 hours.However, we found that endogenous connexin26 expression is affected ifthe culture was maintained for 24 hours. Hence, we reduced the cultureperiod for connexin26 specific oligomers treated corneas to 12 hours,with application of antisense oligomers every 4 hours. Medium waschanged ten minutes prior to every repeat application of antisense orcontrol oligomers. At defined times, corneas were rinsed with PBS,immersed in OCT (Tissue Tek, Japan) and snap-frozen in liquid nitrogen.25 μm cryosections were subsequently cut with a Leica cryostat (CM3050s)and mounted on SuperFrost Plus slides (Menzel, Germany). For both Cx43and Cx26 mRNA analysis corneas were collected 8 hours after a singleantisense treatment.

RNA Isolation and Real-Time PCR

Total RNA was extracted from isolated rat corneas using TRIzol reagent(GIBCO, Invitrogen, USA) according to the manufacturer's protocols. Thequality of RNA samples was assessed by electrophoresis through ethidiumbromide stained agarose gels and the 18S and 28S rRNA bands visualizedunder UV illumination. The extraction yield was quantifiedspectrophotometrically at 260 nm. For real-time PCR, cDNA was preparedfrom 5 ug of total RNA by using oligo dT and superscript II RnaseH-reverse transcriptase (Life Technologies, Invitrogen, USA) in a finalreaction volume of 20 μl. Quantitative PCR reaction was carried out in96-well optical reaction plates using a cDNA equivalent of 100 ng totalRNA for each sample in a volume of 50 μl using the TaqMan Universal PCRMaster Mix (Applied Biosystems, USA) according to the manufacturer'sinstructions. PCR was developed on the ABI PRISM 7700 Sequence Detectionsystem instrument (Applied Biosystems, USA). The thermal cyclingconditions comprised an initial denaturation step at 95° C. for 10minutes and 50 cycles of two-step PCR, including 15 seconds ofdenaturation at 95° C. and 1 minute of annealing-elongation at 60° C.,using the standard protocol of the manufacturer. All experiments wererepeated in triplicate. The monitoring of negative control for eachtarget showed an absence of carryover.

Amplification of 18S rRNA was performed as an internal reference againstwhich other RNA values can be normalized. If the efficiencies of thetarget and 18S rRNA amplifications were approximately equal, then theformula 2^(−ΔΔCt) was used to calculate relative levels of mRNA withoutthe need for a standard curve. If the efficiency of amplification of thetarget and 18SrRNA were significantly different, a relative standardcurve method was used to calculate absolute quantities of mRNA and 18SrRNA for each experiment from the measured Ct, and then the relativemRNA levels of the target gene compared with control quantified afternormalization to 18S rRNA.

All calculations were performed by using PRISM 3.02 software (GraphPad,San Diego). Statistical difference between groups was determined byusing the Student's t test. Comparisons among several groups wereperformed by ANOVA, and significance was calculated by using Dunnett'smultiple comparison test.

Assessment of Antisense Oligomers Efficiency on Blocking Translation

Cy3 and TaqMan (Fam, Tamra) labeled oligomers were used to assesspenetration and stability. Cy3-labeled oligomers (Sigma Genosys) andTaqMan (FAM, TAMRA) labeled oligomers (Applied Biosystems) were appliedwith Pluronic gel to measure both the stability and the penetration ofoligomers into the corneal epithelium. The treated corneas were fixed in4% paraformaldehyde for 20 minutes, mounted in 1% agar and viewed undera 40× water immersion lens as whole mount. The depth of oligomerpenetration was measured using the Z-scan option on a Leica SP2 confocalmicroscope and plots of intensity versus z-distance measured. Thebreakdown of TaqMan oligomers was measured using the Lamdba scan optionon the confocal. Fluorescence resonance energy transfer (or FRET)between the FAM (donor) and TAMRA (receptor) molecule occurs in intact30mer oligomers. When the oligomer is broken down FAM and TAMRA are nolonger in close proximity and FRET no longer occurs.

Immunofluorescent Labelling

Immunolabeling of connexins on corneal sections were performed aspreviously described. In brief, sections were blocked in 10% goat serumand incubated with primary antibody at 1:250 (mouse anti rat connexin43)or 1:500 (rabbit anti rat connexin26) at 4° C. overnight. The sectionswere then washed with PBS, incubated with 1:400 dilution of Alexa 488labeled secondary antibody at room temperature for 2 hours and thenfixed in 4% paraformaldehyde and counterstained with 0.2 μM PropidiumIodide or a 1:50 dilution of Hoechst 33258 for 10 min. Sections weremounted in Citifluor antifade medium (Agarscientific UK). All imageswere collected using either a Leica TCS-4D or Leica SP2 confocal laserscanning microscope and stored as TIF files. All images were collectedusing consistent voltage (520-540 V) and offset (−2) settings. Thevoltage and offset were set using the glow-over-under display option tomaximize the gray scale for images of control tissue. The same settingswere then used for all samples within the same experiment.

For quantification, four optical slices through three micrometers wereprocessed into a single extended focus optical image by using the centerof mass topographic projection option on the TCS-4D. Spots of connexinlabel were counted using NIH image (Scion Corp.) after thresholding at90 to 100 pixel intensity on the 256 grey scale image. The area ofcorneal epithelium was also measured and a connexin density per unitarea was calculated. An average of four extended focus images were usedto calculate the absolute connexin density of each cornea. This numberwas then normalized with the medium connexin density of either sensecontrol treated or gel treated corneas. We have represented the data aspercentage knock down when comparing different treatments.

Deoxyribozymes Selectively Cleave mRNA In Vitro

Sixty six deoxyribozymes were designed specifically against rodentconnexin43 mRNA (Table 5). Twenty two of these deoxyribozymes weredesigned to recognize both mouse and rat connexin43 mRNA. We alsopurchased two defective deoxyribozymes with a single point mutation inthe “10-23” catalytic core as negative controls. The deoxyribozymecleavage results were similar for the rat connexin43 mRNA 1.1 Kb inlength (not shown) and the rat connexin43 mRNA 2.4 Kb in length (FIG.11A). Both rat (FIG. 11A) and mouse (FIG. 11B) connexin43 mRNA appear tohave similar regions accessible to the deoxyribozymes. The resultsindicate four regions on the rodent connexin43 mRNA that are exposed andavailable for deoxyribozyme cleavage. These regions are around 367-466,526-622, 783-885, and 1007-1076 bases from the start ATG codon. The twodefective deoxyribozymes, a1df605 and a1df783, showed no cleavage ofrodent connexin43 mRNA. Deoxyribozymes designed against the 200 basepair 5′ untranslated region of rat connexin43 mRNA also did not show anycleavage activity.

TABLE 5Summary of deoxyribozyme (dz) and antisense (as) oligodeoxynucleotide sequencesshowing various degrees of in vitro and in vivo activity against rat connexin43.in in vivo In vivo Name ODN Sequence 5′ to 3′ vitro protein mRNASEQ ID NO: 32 r43dz14 CCAAGGCA ggctagctacaacga TCCAGTCA − SEQ ID NO: 33a1dz605 CCGTGGGA ggctagctacaacga GTGAGAGG + SEQ ID NO: 34 a1df605CCGTGGGA ggctaActacaacga GTGAGAGG − SEQ ID NO: 35 r43dz769AGTCTTTTG ggctagctacaacga TGGGCTCA − SEQ ID NO: 36 a1dz783TTTGGAGA ggctagctacaacga CCGCAGTC ++ SEQ ID NO: 37 a1df783TTTGGAGA ggctaActacaacga CCGCAGTC − SEQ ID NO: 38 r43dz885DB1ACGAGGAA ggctagctacaacga TGTTTCTG +++ SEQ ID NO: 39 r43dz892TTGCGGC ggctagctacaacga CGAGGAAT − SEQ ID NO: 40 r43dz953CCATGCGA ggctagctacaacga TTTGCTCT +++ SEQ ID NO: 41 r43dz1076TTGGTCCA ggctagctacaacga GATGGCTA +++ SEQ ID NO: 42 DB1GTA ATT GCG GCA GGA GGA ATT GTT TCT + ++ GTC SEQ ID NO: 43 DB1sGACAGAAACAATTCCTCCTGCCGCAATTAC − − SEQ ID NO: 44 r43as14CCAAGGCACTCCAGTCAC − SEQ ID NO: 45 a1as605 TCCGTGGGACGTGAGAGGA ++ ++SEQ ID NO: 46 r43as769 AGTCTTTTGATGGGCTCA up up SEQ ID NO: 47 a1as783TTTTGGAGATCCGCAGTCT + ++ SEQ ID NO: 48 r43as885 CACGAGGAATTGTTTCTGT +SEQ ID NO: 49 r43as892 TTTGCGGCACGAGGAATT − SEQ ID NO: 50 a1as953CCCATGCGATTTTGCTCTG + SEQ ID NO: 51 a1as1076 GTTGGTCCACGATGGCTAA +

Table 5 shows those ribozyme and antisense sequences selected on thebasis of in vitro ribozyme cleavage studies for in vivo analysis (mRNAand/or protein levels) or where defective ribozyme controls (SEQ IDNO:56 and SEQ ID NO:59) are compared with normal ribozymes.

The oligomer names have the prefix r43 where they are specific only torat connexin43 only; the prefix a1 denotes specificity against bothmouse and rat. All oligomer sequences are unmodified phosphodiesteroligodeoxynucleotides. “ggctagctacaacga” represents the “10-23”catalytic core of the deoxyribozymes and “ggctaActacaacga” is thedefective mutant control. DB1 is a 30-mer version of as885 (marked inlower case) and DB1s is the sense control of DB1 sequence. In vitroeffects were measured as percentage mRNA cleavage by individualdeoxyribozymes. In vivo effects were measured by immunolabeling ofconnexin43 in corneal sections (refer to FIG. 15) or Real-Time PCRassessment of surviving mRNA levels (refer to FIG. 16). +++ means >75%,++ means between 50% to 75%, + means between 25% to 50%, and − meansbetween 0% to 25% in vitro cleavage of mRNA or in vivo reduction ofprotein and mRNA expressions. up means an increase in connexin43 proteinexpression when compared to DB 1 sense or gel only control treatment.

We also tested forty four deoxyribozymes designed specifically againstrodent connexin26 mRNA (Table 6), of which 17 deoxyribozymes match bothmouse and rat connexin26 mRNA. The rat connexin26 mRNA appeared as adouble band on the gel owing to the presence of two T7 RNA polymerasepromotors on the cloning plasmid. The cleavage results show thatconnexin26 mRNA has at least two regions accessible to deoxyribozymes,in the 318-379 and 493-567 base regions (FIG. 12A, 12B). These figuresshow that most consistently cleaving deoxyribozyme is the cx26dz330,which cleaves both species of mRNA within one hour. The two defectivedeoxyribozymes (b2df351 and b2df379) showed no cleavage of rodentconnexin26 mRNA. The deoxyribozymes cx26dz341, dz351, dz375, dz379consistently cleave rat connexin26 mRNA at a higher rate compared tomouse connexin26 mRNA. On the other hand, mcx26dz153 and dz567 appear tobe superior connexin26 deoxyribozymes in mouse when compared to rat.

TABLE 6Deoxyribozyme (dz) and antisense (as) oligodeoxynucleotide sequences showingvarious degrees of in vitro and in vivo activity against rodent connexin26.in in vivo In vivo Name ODN Sequence 5′ to 3′ vitro protein mRNASEQ ID NO: 52 m26dz153 GTTGCAGA ggctagctacaacga AAAATCGG +++SEQ ID NO: 53 b2dz330 GTTCTTTA ggctagctacaacga CTCTCCCT +++SEQ ID NO: 54 b2dz341 GTCCTTAAA ggctagctacaacga TCGTTCTTT +++SEQ ID NO: 55 b2dz351 TCTCTTCGA ggctagctacaacga GTCCTTAAA +++SEQ ID NO: 56 b2df351 TCTCTTCGA ggctaActacaacga GTCCTTAAA −SEQ ID NO: 57 b2dz375 GATACGGA ggctagctacaacga CTTCTGGG +++SEQ ID NO: 58 b2dz379 CTTCGATA ggctagctacaacga GGACCTTC +++SEQ ID NO: 59 b2df379 CTTCGATA ggctaActacaacga GGACCTTC − SEQ ID NO: 60m26dz567 GGTGAAGA ggctagctacaacga AGTCTTTTCT +++ SEQ ID NO: 61 b2as330nCCTTAAACTCGTTCTTTATCTCTCCCTTCA − ++ SEQ ID NO: 62 b2rv330nACTTCCCTCTCTATTTCTTGCTCAAATTCC − − SEQ ID NO: 63 r26as375nTACGGACCTTCTGGGTTTTGATCTCTTCGA − + SEQ ID NO: 64 r26rv375nAGCTTCTCTAGTTTTGGGTCTTCCAGGCAT −

Table 6 shows those ribozyme and antisense sequences that consistentlycleaved the mRNA in vitro, were selected on the basis of in vitroribozyme cleavage studies for in vivo analysis (mRNA and/or proteinlevels), or where used as defective ribozyme controls. The oligomernames have the prefix m26 or r26 where they are specific only to mouseor rat connexin26 mRNA respectively, and the prefix b2 denotesspecificity against both species. All oligomer sequences are unmodifiedphosphodiester oligodeoxynucleotides. “ggctagctacaacga” represents the“10-23” catalytic core of the deoxyribozymes and “ggctaActacaacga” isthe defective mutant control. A reverse control (rv) was also used tocontrol for any non-specific effects of antisense oligomers. In vivoeffects were measured by immunolabeling of connexin26 in cornealsections and Real-Time PCR of the target mRNA expression (refer to FIG.17). +++ means >75%, ++ means between 50% to 75%, + means between 25% to50%, and − means between 0% to 25% in vitro cleavage of mRNA or in vivoreduction of protein and mRNA expressions.

Fluoresecently Labeled ODN in Pluronic Gel can Penetrate the CornealEpithelium

Rat corneas maintain expression of both connexin43 and connexin26 inorgan culture and are easily accessible to the delivery of antisenseoligomers by 30% Pluronic F-127 gel. The rat cornea organ culture wastherefore selected as the model system to test the effectiveness of theantisense oligodeoxynucleotides designs derived from the in vitro model.We cultured rat corneas for 24 hours and found that the endotheliumremains intact. However culture times longer than 48 hours appeared toaffect the opacity of the corneas and were therefore not used. Cy3labeled oligomers were used to determine the extent of penetration ofthe oligomer into the cultured cornea. Confocal optical slices downthrough the intact cornea show that fluorescent signal is present withCY3 labeled oligomers at FIG. 13A shows fluorescent signal 10 μm deep inthe cultured cornea 1 hour after initial treatment. TaqMan probesconjugated to oligodeoxynucleotides were used to measure and demonstratethe delivery of intact oligodeoxynucleotide with 30% Pluronic gel intocorneal epithelium. A significant proportion of oligomer remained intactone hour after treatment (FIG. 13B, 13C). The punctuate signal of intactoligomers (FRET occurring in FIG. 13C) can be seen as the red(represented on gray-scale) wavelength while signal from degradedoligomers (no FRET) appears in the green (represented on gray-scale)emission spectrum (FIG. 13B).

Deoxyribozyme Assay Predicts ODNs that can Knockdown Connexin43 Proteinin Corneal Epithelium.

In a preliminary experiment, we treated rat corneas with a singleapplication of our positive control, DB1, and found no significantchanges in connexin43 protein expression after 8 hours. Clear proteinknockdown at 24 hours was seen after three applications at eight hourlyintervals. Based in part on results from the deoxyribozyme cleavageassay we tested certain antisense oligomers in vivo (DB1, r43as605,r43as783, r43as885, r43as953 and r43as1076), as well as antisenseoligomers that were predicted to be non-functional (r43as14, r43as769and r43as892), and a negative control (DB1 sense). We found knockdown ofconnexin43 protein levels after 24 hours of treatment compared tocontrols (FIG. 14A) with all of the antisense oligomers that we haddetermined should be positive (FIG. 14C, 14E, 14G). All three of thosepredicted to be negative, and the negative control oligomer, did notaffect connexin43 expression (FIG. 14B, 14D, 4F, 4H). DB1, a 30-merversion of as885, showed a similar percentage knock down to the shorteras885 (just under 50% knockdown). One of the better antisense oligomersidentified in this experiment appeared to be as605 with a 64% reductionin protein level. A summary of these results quantified is presented inFIG. 15.

To test the technique for other connexins, further oligodeoxynucleotideswere designed and tested for connexin26. Two 30-mer antisenseoligodeoxynucleotides designated as r26as330N and 375N, together withtheir appropriate reverse control oligodeoxynucleotides were designedagainst connexin26 based on regions within the cleavage areas of b2dz330and b2dz375. We found that these antisense oligodeoxynucleotides (as330Nand as375N) did not, however, lead to a significant difference inprotein expression levels within the 12 hour time period for theseexperiments when antisense oligomers treated cultures were compared withthe reverse control treated corneas.

Antisense ODNS Lead to Reduction in Connexin43 and Connexin 26 mRNA

Real time PCR was used to determine the effect of antisenseoligodeoxynucleotides on mRNA levels. It confirmed that antisenseoligodeoxynucleotides that knock down connexin43 protein expression(as605, as885, DB1) also have lower connexin43 mRNA levels compared tocontrol corneas within 8 hours after treatment (FIG. 16). The percentagereduction in relative levels of connexin43 mRNA correlated well with thelevel of reduction of connexin43 protein. The negative antisenseoligomer (as769) and negative controls (DB1 sense, gel only) exhibitedunchanged levels of connexin43 mRNA compared to control corneas.

Connexin26 mRNA expression was also significantly reduced by as330N andas 375N within 8 hours of antisense treatment (FIG. 17). The reversesequence control for as330N and a gel only control exhibited no effecton mRNA levels.

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe levels of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “consisting essentially of”, and“consisting of” may be replaced with either of the other two terms inthe specification. Also, the terms “comprising”, “including”,containing”, etc. are to be read expansively and without limitation. Themethods and processes illustratively described herein suitably may bepracticed in differing orders of steps, and that they are notnecessarily restricted to the orders of steps indicated herein or in theclaims. It is also that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Under no circumstances may thepatent be interpreted to be limited to the specific examples orembodiments or methods specifically disclosed herein. Under nocircumstances may the patent be interpreted to be limited by anystatement made by any Examiner or any other official or employee of thePatent and Trademark Office unless such statement is specifically andwithout qualification or reservation expressly adopted in a responsivewriting by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is: 1-56. (canceled)
 57. A method of treating an injuryto the central nervous system, the method comprising administering anantisense compound to a site proximal to a preexisting wound of thecentral nervous system in association with a surgical procedureperformed on a subject to treat said injury to the central nervoussystem, wherein said antisense compound is targeted to at least about 8nucleobases of a nucleic acid molecule encoding a connexin having anucleobase sequence selected from SEQ ID NO:12-31.
 58. The method ofclaim 57 wherein said injury to the central nervous system is a spinalcord injury.
 59. The method of claim 57 wherein said antisense compoundis administered to a subject at least 24 hours after a physical traumato the spinal cord.
 60. The method of claim 57 wherein said antisensecompound is administered in conjunction with a procedure to graft nervetissue into a spinal cord injury region of a subject.
 61. The method ofclaim 57 wherein said antisense compound decreases scar formation. 62.The method of claim 57 wherein said antisense compound reducesinflammation.
 63. The method of claim 57 wherein said antisense compoundpromotes wound healing.
 64. The method of claim 57 used in associationwith a surgical implantation procedure.
 65. The method of claim 57wherein said antisense compound is directed to connexin 43 and isadministered to regulate epithelial basal cell division and growth. 66.The method of claim 57 wherein said antisense compound is directed toconnexin 31.1 and is administered to regulate outer layerkeratinisation.
 67. Use of an antisense compound in the preparation of amedicament for reducing tissue damage associated with an ophthalmicprocedure, wherein said antisense compound inhibits the expression of aconnexin protein in the eye or in cells associated with the eye of asubject.
 68. Use of an antisense compound in the preparation of amedicament for tissue engineering in association with an ophthalmicprocedure, wherein said antisense compound inhibits the expression of aconnexin protein in the eye or in cells associated with the eye of asubject.